WO2024235355A1 - Chimeric antigen receptor, recombinant immune cell and use - Google Patents

Abstract

A recombinant immune cell, wherein the recombinant immune cell comprises: (i) one or more structures for cell adoptive therapy; and (ii) a gene regulatory system capable of reducing or removing the expression and/or function of the BCOR gene and the ZC3H12A gene in an immune cell; wherein the structure for cell adoptive therapy specifically binds to an antigen derived from eosinophils.

Description

Chimeric antigen receptor, recombinant immune cell and use thereof

Priority and related applications

The present disclosure claims the priority of Chinese patent application 202310550006.5 filed on May 16, 2023 and entitled “A Chimeric Antigen Receptor, Recombinant Immune Cell and Uses”, and all the contents of the application including the appendix are incorporated into the present disclosure as a reference.

Technical Field

The present disclosure belongs to the field of cell technology and relates to chimeric antigen receptors, recombinant immune cells and uses. More specifically, the present disclosure relates to the preparation of chimeric receptor T cells based on IL-5 molecules that simultaneously inhibit or knock out ZC3H12A and BCOR genes, and using them as cell carriers to release IL-4 mutant proteins with inhibitory functions, and ultimately construct multi-target targeted cell drugs with long-term therapeutic, curative and preventive effects.

Background Art

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 a very significant effect in the immunotherapy of tumors, especially for lymphocytic leukemia. At the same time, more and more evidence shows that cell immunotherapy will also play an important role in the treatment of autoimmune diseases.

As we all know, allergic asthma is a typical allergic disease mediated by type 2 immune response, which leads to disturbance of local immune homeostasis, specifically manifested as a significant increase in eosinophils, and ultimately leads to damage and dysfunction of solid organs. However, the continued enhancement of type 2 immune response further strengthens the course of the disease. Although many chemical drugs and antibody drugs targeting different disease processes have been launched one after another, the gradual resistance of patients to the corresponding drugs still casts a shadow on the treatment of the disease. Cell therapy has obviously become one of the more efficient and ideal treatment options. However, unlike cell immunotherapy for tumors, the efficacy of CAR-T therapy can be enhanced by pretreatment with chemotherapy drugs. For the treatment of inflammatory diseases or allergic diseases mediated by type 2 immune response and diseases with eosinophils as effector cells, this approach will undoubtedly cause unnecessary and additional harm to patients. Therefore, although there are many CAR molecules targeting different targets that show the expected effect of killing target cells in vitro, once these CAR molecule-modified T cells are adoptively transferred into the body, they show major defects, neither amplifying nor killing target cells. This situation also leads to the inability to obtain long-term cure for type 2 immune response-mediated inflammatory diseases or allergic diseases, including cellular immunotherapy for allergic asthma.

The 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 the IL-5CAR-T cells were injected into mice, asthma was induced in the mice, and IL-5CAR-T was observed to have a protective effect on asthma in mice, and compared with the control group without IL-5CAR-T cells, a decrease in eosinophils was still observed at 3 months. However, the cited literature 1 only proves 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 inflammatory diseases or allergic diseases mediated by type 2 immune responses and diseases with eosinophils as effector cells.

Therefore, cell adoptive therapy for inflammatory diseases or allergic diseases mediated by type 2 immune responses and diseases with eosinophils as effector cells still needs to be further developed.

Summary of the invention

Problem that the invention aims to solve

In response to the above-mentioned problems existing in the prior art, the present disclosure provides a chimeric antigen receptor, recombinant immune cells, related biomaterials, compositions and uses. Among them, the recombinant immune cells edit T cells by means of gene knockout or gene suppression, so that they can effectively target disease-related eosinophils in the body for a long time through chimeric receptors based on IL-5 molecules; and use this as a carrier to release inhibitory IL-4 mutant proteins to effectively inhibit type 2 immune responses. Ultimately, in multiple disease models, this type of gene-knocked multi-target targeted recombinant cells showed long-term and efficient treatment, cure and prevention of diseases.

Solutions for solving problems

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

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

(ii) a gene regulatory system capable of reducing or eliminating the expression and/or function of the BCOR gene and the ZC3H12A gene in immune cells;

Wherein, the structure for adoptive cell therapy specifically binds to antigens derived from eosinophils.

[2] The recombinant immune cell according to [1], wherein the eosinophil-derived antigen comprises one or more of IL-5Rα, CRTh2, CCR3, and Siglec-8;

Preferably, the eosinophil-derived antigen is IL-5Rα.

[3] The recombinant immune cell according to [1] or [2], wherein the structure for adoptive cell therapy is selected from one or more of a chimeric antigen receptor, a T cell antigen receptor, a receptor based on ligand receptor binding, and a synthetic T cell receptor and antigen receptor;

Preferably, the structure for adoptive cell therapy is a chimeric antigen receptor, which comprises:

(a) a polypeptide derived from IL-5;

(b) a polypeptide derived from CD28; and,

(c) A polypeptide derived from CD3zeta.

[4] The recombinant immune cell according to [3], wherein

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,

The amino acid sequence of the polypeptide derived from CD28 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.

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

( _a1 ) the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16;

( _a2 ) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:16, and that has or partially has the function of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:16;

( _a3 ) an amino acid sequence in which one or more amino acid residues are truncated, added, substituted, deleted or inserted into the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16, and which has or partially has the function of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16; or

( _a4 ) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16 under stringent conditions, and the amino acid sequence has or partially has the function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16, and the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

The recombinant immune cells provided by the present disclosure have the expression and/or function of the BCOR gene and the ZC3H12A gene reduced or eliminated, and are applied to T cells modified with IL-5CAR to enable them to have or enhance in vivo activity.

[6] The recombinant immune cell according to any one of [1] to [5], wherein the recombinant immune cell further comprises:

(iii) biomolecules for the treatment of diseases;

Optionally, the biological molecule for treating a disease is selected from the group consisting of: cytokines, hormones, growth factors, coagulation factors, chemokines, co-stimulatory molecules, activation peptides, antibodies or antigen-binding fragments thereof, or mutants thereof;

Preferably, the biological molecule for treating a disease is selected from one or more of 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;

More preferably, the biological molecule for treating the disease is an IL-4 mutant, whose amino acid sequence is shown in SEQ ID NO: 7 or SEQ ID NO: 20.

[7] The recombinant immune cell according to any one of [1] to [6], wherein the immune cell is derived from a mammal;

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

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

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-δT cells, α-βT cells, CD4-CD8- double negative T cells or NKT cells.

[8]. The recombinant immune cell according to any one of [1] to [7], wherein the gene regulation system uses gene knockout technology, gene silencing technology, inactivation mutation technology, PROTAC technology or small molecule inhibitors to treat the BCOR gene and ZC3H12A gene in the recombinant immune cell.

[9] A chimeric antigen receptor comprising:

(a) a polypeptide derived from IL-5;

(b) a polypeptide derived from CD28; and,

(c) a polypeptide derived from CD3zeta;

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

( _a1 ) the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16;

( _a2 ) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16, and that has or partially has the function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16;

( _a3 ) an amino acid sequence in which one or more amino acid residues are truncated, added, substituted, deleted or inserted into the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16, and which has or partially has the function of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16; or

( _a4 ) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16 under stringent conditions, and the amino acid sequence has or partially has the function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16, and the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

The present invention provides a new CAR molecule, which uses the full length of the IL-5 molecule as the target recognition structure and combines CD28 as the proximal membrane end as the extracellular segment, uses the transmembrane end of the CD28 molecule as the transmembrane region of the CAR, and combines the CD28 intracellular segment and the CD3zeta intracellular segment as the intracellular region of the CAR signal transduction, ultimately constructing a CAR molecule that can effectively recognize target cells and induce downstream activation signals.

[10]. A biomaterial, wherein the biomaterial comprises at least one of the following _b1 ) to _b3 ):

_b1 ) a polynucleotide encoding the chimeric antigen receptor as described in [9];

_b2 ) a vector containing the polynucleotide described in _b1 );

_b3 ) A cell containing the vector described in _b2 ).

[11]. A composition comprising the recombinant immune cell described in any one of [1] to [8], the chimeric antigen receptor described in [9] and/or the biomaterial described in [10]; and, optionally, a pharmaceutically acceptable carrier.

[12] Use of the recombinant immune cell described in any one of [1] to [8], the chimeric antigen receptor described in [9] and/or the biomaterial described in [10] in the preparation of a medicament for treating and/or preventing a disease or condition;

Wherein, the disease or condition is selected from inflammatory diseases or allergic diseases mediated by type 2 immune response, and diseases with eosinophils as effector cells;

Optionally, the inflammatory disease or allergic disease mediated by the type 2 immune response includes one or more of asthma, allergic rhinitis, inflammatory skin disease, and food allergy;

Optionally, the diseases with eosinophils as effector cells include one or more of acute and chronic asthma, eosinophilia, nasal polyps caused by eosinophils, enteritis caused by eosinophils, eosinophilic dermatitis, chronic obstructive pulmonary disease, and eosinophilic leukemia.

[13]. Use of the recombinant immune cell described in any one of [1] to [8] as a carrier for delivering biological molecules for treating diseases.

The recombinant immune cells disclosed herein are used as carriers to express different types of molecules with different activities, including but not limited to the expression of IL-4 mutant proteins with inhibitory functions. It also includes the expression of proteins with physiological activity, therapeutically active antibodies, CAR molecules or TCR molecules with other targeting capabilities, cell surface molecules with targeting capabilities, and molecules with the function of regulating intracellular cell transduction and transcription.

[14] The method for preparing a recombinant immune cell according to any one of [1] to [8], comprising:

(i) a step of introducing a construct for adoptive cell therapy into immune cells; and,

(ii) a step of introducing a gene regulatory system into immune cells;

and, optionally,

(iii) A step of introducing biomolecules for treating diseases into immune cells.

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

Effects of the Invention

The experiments disclosed herein prove that the CAR-T cells with double knockout of ZC3H12A/BCOR obtained by gene editing in the present disclosure enable the IL-5CAR-modified T cells that do not have in vivo cell activity to be truly applied in vivo, showing the following very obvious advantages:

1) Avoid using pretreatments with extremely toxic side effects;

2) It can effectively eliminate target cells, namely eosinophils, for a long time. The gene-edited IL-5CAR-T cells exist in the body for a long time, which is equivalent to a group of cells being implanted in the body for a long time, solving the problem of the long-term effectiveness of CAR-T treatment;

3) A single treatment can achieve the effect of prevention and long-term cure. The results of animal experiments have shown that, whether in acute or chronic asthma models, recombinant immune cells have shown obvious therapeutic effects in completely clearing inflammatory granulocytes, reshaping the local immune environment, and improving the function of lung parenchymal organs; at the same time, they provide the body with the ability of immune tolerance, which can effectively prevent the strong stimulation of allergens.

4) These long-term IL-5CAR-T cells in the body can also be used as carriers to secrete therapeutic proteins, including antibodies, peptides, and hormones. For example, the expression of IL-4 mutant protein with inhibitory function can significantly inhibit type 2 immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Recombinant IL-5CAR-T cells with simultaneous knockout of Bcor and Zc3h12a persist in vivo for a long time and eliminate 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, IL-5CAR-T cells killing MC-38 tumor cells expressing IL-5Ra in vitro; D, Statistical results of IL-5CAR-T cell killing ability at different effector cell-target cell ratios; E, Statistical results of IL-5CAR-T cells killing eosinophils; F, Statistical results of IL-5CAR-T cells killing eosinophils at 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 flow cytometry analysis of the ratio of peripheral blood CAR-T cells and eosinophils on the 14th day; H is the statistical results 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 is the proportion of peripheral blood CAR-T cells analyzed by flow cytometry at different time points after 5T _IF was adoptively transferred into recipient mice; J is the proportion of peripheral blood eosinophils analyzed by flow cytometry at different time points after 5T _IF was adoptively transferred into recipient mice; 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.

Figure 2. IL-5CAR-T cells neither expand nor kill target cells after adoptive transfer into mice without pretreatment. Among them, A is a schematic diagram of the experimental process; B is the flow cytometric analysis of the proportion of CAR-T cells in mouse peripheral blood cells to CD8 ⁺ T cells on the 7th day after IL-5CAR-T cell adoptive transfer; C is the flow cytometric analysis of the proportion of eosinophils in mouse peripheral blood cells on the 7th day after IL-5CAR-T cell adoptive transfer; D is the statistical analysis result of B and C, n=4, data are mean±SEM, unpaired student's t-test: NS, no significant difference; E is the flow cytometric analysis of the proportion of CAR-T cells in peripheral blood to CD8 ⁺ T cells and the proportion of eosinophils in peripheral blood on the 7th day and 28th day after adoptive transfer of different doses of IL-5CAR-T cells; F and G are the statistical analysis results of the results of E; H is the flow cytometric analysis of the proportion of CAR-T cells in the spleen and bone marrow to CD8 ⁺ The proportion of T cells and eosinophils; I and J are statistical analysis results of H; F, G, I, J statistical analysis, n = 4, data are mean ± SEM, one-way ANOVA: NS, no significant difference.

Figure 3. 5T _IF cells can be used as carriers to secrete IL-4 mutant proteins with inhibitory functions, thereby inhibiting type 2 immune responses. Among them, A is a schematic diagram; B is the statistical results of ELISA detection of the content of IL-4 mutant proteins secreted by 5T _IF 4 cells in the supernatant of in vitro culture and ELISA detection of the content of IL-4 mutant proteins secreted in the serum of recipient mice adoptively transferred with 5T _IF 4 cells at steady state; C is a schematic diagram of the experimental process; D is the statistical results of ELISA detection of IL-13 content in the serum of immunized mice; E is the statistical results of ELISA detection of total IgE content in the serum of immunized mice; F is a representative result of flow cytometry analysis of plasma cell levels in the spleen of immunized mice; G is the statistical results of 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.

Figure 4. 5T _IF 4 cells have the same long-term target cell killing function as 5T _IF cells. Among them, A is a flowchart for constructing 5T _IF 4 cells; B is a representative figure for flow cytometry analysis of IL-5CAR molecular membrane expression levels; C is the statistical analysis results of flow cytometry analysis of IL-5CAR membrane efficiency and expression levels, n = 3, data are mean ± SEM, using unpaired student's t-test: NS, no significant difference; D is a representative figure of flow cytometry analysis of the proportion of CAR-T cells in peripheral blood cells to CD8 ⁺ T cells 2 weeks after adoptive transfer; E is a representative figure of flow cytometry analysis of the proportion of eosinophils in peripheral blood cells 2 weeks after adoptive transfer; F is the statistical analysis results of D and E, n = 4, data are mean ± SEM, using one-way ANOVA: NS, no significant difference; **p < 0.01.

Figure 5. 5T _IF cells and 5T _IF 4 cells can cure acute allergic asthma induced by OVA antigen. A is the flow chart of the experiment; B is a representative diagram of the proportion of CAR-T cells in the lungs to CD8 ⁺ T cells by flow cytometry; C is the statistical analysis results of the proportion of CAR-T cells in the lungs to CD8 ⁺ T cells and the absolute number of CAR-T cells by flow cytometry; D is the statistical analysis results of the HE staining results; G and H are the statistical results of the absolute number of various blood cells in the lung lavage fluid and the lungs by flow cytometry, among which CD45 ⁺ is all white blood cells, EOS is eosinophils, NEU is neutrophils, MAC is macrophages, T is T cells, and B is B cells; E is the statistical result of ELISA detection of IL-13 content in lung lavage fluid; F is the statistical result of ELISA detection of total IgE content in serum; C, E, F, G, H statistical analysis, n = 4, D statistical analysis, n = 8, data are mean ± SEM, using one-way ANOVA: NS, no significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6. 5T _IF cells and 5T _IF 4 cells can cure OVA antigen-induced chronic long-term allergic asthma. Among them, A is the flowchart of the experiment; B is a representative figure of the proportion of CAR-T cells to CD8 ⁺ T cells in the lungs by flow cytometry; C is the statistical analysis results of the proportion of CAR-T cells to CD8 ⁺ T cells in the lungs and the absolute number of CAR-T cells by flow cytometry; D is the statistical analysis results of HE staining results and PAS staining results; G and H are the statistical results of the absolute number of various blood cells in lung lavage fluid and lungs detected by flow cytometry; E is the statistical result of ELISA detection of IL-13 content in lung lavage fluid; F is the statistical result of ELISA detection of total IgE content in serum; statistical analysis of C, E, F, G, 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.

Figure 7. 5T _IF cells and 5T _IF 4 cells prevent IL-33-induced asthma. A is the flow chart of the experiment; B is a representative figure of the proportion of CAR-T cells in the lungs to CD8 ⁺ T cells by flow cytometry; C is the statistical analysis results of the proportion of CAR-T cells in the lungs to CD8 ⁺ T cells and the absolute number of CAR-T cells by flow cytometry; D is the statistical analysis results of HE staining results; E is the statistical result of ELISA detection of IL-13 content in lung lavage fluid; F and G are the statistical results of flow cytometry detection of the absolute number of various blood cells in lung lavage fluid and lungs; C, E, F, G statistical analysis, n = 4, D statistical analysis, 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.

Figure 8.5T _IF cells and 5T _IF 4 cells prevent HDM-induced allergic asthma. A is the flow chart of the experiment; B is a representative figure of the proportion of CAR-T cells in the lungs to CD8 ⁺ T cells by flow cytometry; C is the statistical analysis results of the proportion of CAR-T cells in the lungs to CD8 ⁺ T cells and the absolute number of CAR-T cells by flow cytometry; D is the statistical analysis results of HE staining results; E is the statistical result of ELISA detection of IL-13 content in lung lavage fluid; F and G are the statistical results of flow cytometry detection of the absolute number of various blood cells in lung lavage fluid and lungs; C, E, F, G statistical analysis, n = 4, D statistical analysis, 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.

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

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

Figure 11. Schematic diagram of the principle of 5T _IF 4 curing and preventing diseases. Cells carrying IL-5CAR molecules and lacking ZC3H12A and BCOR, and expressing IL-4 mutants at the same time, 5T _IF 4, can completely kill eosinophils, while inhibiting type 2 immune responses mediated by IL-4 and IL-13, and ultimately cure or prevent diseases mediated by type 2 immune responses and diseases related to eosinophilia.

DETAILED DESCRIPTION

Before the present disclosure is further described, it is to be understood that the present disclosure is not limited to the particular embodiments described herein; it is also to be understood that the terminology used herein is for the purpose of describing only and not limiting the particular embodiments.

[Definition of terms]

In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values A and B.

In the present specification, the use of “substantially” or “essentially” means that the standard deviation from a theoretical model or theoretical data is within a range of 5%, preferably 3%, and more preferably 1%.

In this specification, the word "may" means both performing a certain process and not performing a certain process.

In the present specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes cases where the event occurs and cases where it does not occur.

In this specification, the references to "some specific/preferred embodiments", "other specific/preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and/or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

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

According to the present disclosure, the terms "polypeptide", "protein" and "peptide" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with similar peptide backbones.

According to the present disclosure, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. 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.

In accordance with the present disclosure, the three letter codes and one letter codes for amino acids used are as described in J. biol. chem, 243, p3558 (1968).

According to the present disclosure, amino acid "addition" refers to the addition of amino acids at the C-terminus or N-terminus of an amino acid sequence. According to the present disclosure, amino acid "deletion" refers to the deletion of 1, 2 or 3 or more amino acids from an amino acid sequence. According to the present disclosure, amino acid "insertion" refers to the insertion of amino acid residues at appropriate positions in an amino acid sequence, and 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.

According to the present disclosure, amino acid "substitution" refers to the replacement of a certain amino acid residue at a certain position in an amino acid sequence by another amino acid residue; wherein the "substitution" may be a conservative amino acid substitution.

According to the present 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, etc.), so that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art know that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224, (4th ed.)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity. Exemplary conservative substitutions are set forth below in "Exemplary Amino Acid Conservative Substitutions".

Exemplary conservative amino acid substitutions

"Identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent 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 × 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is performed when the two sequences are aligned to obtain the maximum percent identity.

According to the present disclosure, "moderate to very high stringency conditions" include "moderate stringency conditions", "moderate-high stringency conditions", "high stringency conditions" or "very high stringency conditions", which describe conditions for nucleic acid hybridization and washing. Guidance for conducting hybridization reactions is found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in this document, and either can be used. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions are in 6× sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2×SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the washing temperature can be increased to 55°C); (2) moderate stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C and preferably; (4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2×SSC, 1% SDS at 65°C.

"Administering," "giving," and "treating" when applied to an animal, a human, an experimental subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administering," "giving," and "treating" may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic agent, a binding composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or a research subject, refers to therapeutic, prophylactic or preventative measures, research, and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent, such as a recombinant immune cell comprising the present disclosure, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, either by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical test method commonly used by physicians or other health care professionals to evaluate the severity or progression of the symptom.

In this specification, the term "prevention" refers to the preventive treatment of subjects who do not have a disease now or in the past but are at risk of developing a disease or who have had a disease in the past and do not have a disease now but are at risk of disease recurrence. In certain embodiments, the subject has a higher risk of developing a disease or a higher risk of disease recurrence compared to the average healthy member of the subject population.

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

In this specification, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with a condition. A therapeutically effective amount refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" can include an amount that improves overall therapy; reduces or avoids symptoms, signs, or causes of a condition; and/or enhances the therapeutic efficacy of another therapeutic agent.

In this specification, a "prophylactically effective amount" is an amount sufficient to prevent a condition or one or more symptoms associated with a condition or to prevent its recurrence. A prophylactically effective amount refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a condition. The term "prophylactically effective amount" may include an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent.

In this specification, "pharmaceutical composition" or "composition" means containing one or more recombinant immune cells described herein, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

In this specification, the term "pharmaceutically acceptable" (or "pharmacologically acceptable", "pharmaceutically usable") refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other undesirable reactions when administered to animals or humans, as appropriate. The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic agents and absorption delaying agents, buffers, excipients, adhesives, lubricants, gels, surfactants, etc. that can be used as media for pharmaceutically acceptable substances.

"BCOR gene" and "Bcor gene" can be used interchangeably unless otherwise specified. The "BCOR gene" is the BCOR gene of any target subject.

"ZC3H12A gene" and "Zc3h12a gene" can be used interchangeably unless otherwise specified. The "ZC3H12A gene" is the "ZC3H12A gene" of any target subject.

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

"Immortal-like and Functional" or "Immortal-like" (abbreviated as IF) refers to the characteristics of cells that acquire the ability to continue to grow and proliferate, and have no phenotypic characteristics of malignant transformation, no tumorigenicity, and no invasiveness and metastasis of tumor cells. In this article, the subscript IF is used to represent T cells with "immortal-like" properties that have knocked out Bcor and Zc3h12a at the same time, referred to as T _IF , including 5T _IF , 4T _IF 4.

"Subject" or "host" refers to human or non-human animals, including mammals. For example, primates (such as humans, monkeys), cattle, sheep, goats, alpacas, horses, dogs, cats, rabbits, rats, mice, etc. "Subject" or "host" includes therapeutic and non-therapeutic types. "Subject" or "host" includes experimental animal models or animals used to produce biological molecules expressing therapeutic diseases, i.e., "non-therapeutic hosts" or "non-therapeutic subjects".

“Related molecules for treating diseases” or “biological molecules for treating diseases”, unless otherwise specified, refer to related molecules or biological molecules that are introduced into immune cells via “foreign genes” and secreted by recombinant immune cells.

As used herein, "pretreatment" refers to the advance treatment of patients or experimental animals before cell input. Conventional CAR-T therapy and other cell therapies require chemotherapy or radiotherapy to remove lymphocytes in the patient's body to provide space and other factors for the input cells. Without pretreatment, the input T cells proliferate insufficiently and the efficacy is limited. However, pretreatment can cause many side effects, including immunodeficiency and cytokine storm. The 5T _IF and 4T _IF 4 cells prepared in the present disclosure can be expanded in vivo without any pretreatment, kill target cells, and persist for a long time.

[Detailed description of the invention]

The present disclosure found that T cells modified by chimeric receptor molecules constructed with IL-5 molecules as recognition structures can target and kill tumor cell lines expressing IL-5α receptor molecules, and can target and kill eosinophils isolated from mice. However, T cells modified by this CAR molecule cannot proliferate and kill target cells in vivo.

Based on this discovery, the present disclosure provides a recombinant immune cell and a preparation method thereof, a method for treating asthma, a method for preventing the disease, and extends to a method for treating inflammatory diseases or allergic diseases mediated by type 2 immune response, and a method for treating diseases with eosinophils as effector cells. By reducing or eliminating the expression and/or biological functions of the BCOR gene and the ZC3H12A gene, the T cells modified by the IL-5CAR molecule can be expanded in vivo without pretreatment, persist and exert a killing function for a long time, giving the recombinant immune cells extremely strong stemness or functional immortality (Immortal-like and Functional) characteristics. At the same time, by making it express IL-4 mutant protein, the scope and indications of its disease treatment are expanded, so that it can effectively inhibit type 2 immune response and ultimately be used for the treatment of the disease. In acute and chronic asthma models, the recombinant immune cells showed significant disease treatment and healing effects. At the same time, it showed a significant therapeutic effect in inhibiting type 2 immune response. The recombinant immune cells provided by the present disclosure only need to be injected into the recombinant cells once, and asthma and other diseases caused by eosinophilia and type 2 inflammation can be cured for life. At the same time, in the allergen exposure model, the recombinant immune cells endowed the body with significant ability to resist airway allergy reactions and had a preventive effect.

<Chimeric Antigen Receptor (CAR)>

In some aspects of the disclosure, the present disclosure provides a chimeric antigen receptor comprising:

(A) an extracellular domain that specifically binds to an eosinophil-derived antigen;

(B) transmembrane domain;

(C) Intracellular signaling domain.

In some specific embodiments, the present disclosure provides a chimeric antigen receptor comprising:

(a) a polypeptide derived from IL-5;

(b) a polypeptide derived from CD28; and,

(c) A polypeptide derived from CD3zeta.

(Extracellular domain)

In some embodiments, the antigen derived from eosinophils includes one or more of IL-5Rα, CRTh2, CCR3, Siglec-8. In some embodiments, the antigen derived from eosinophils includes IL-5Rα. In some more preferred embodiments, the antigen derived from eosinophils is IL-5Rα.

In some embodiments, the extracellular domain comprises a polypeptide derived from cytokine interleukin 5 (IL-5), which is capable of specifically binding to IL-5Rα. In some specific embodiments, the extracellular domain comprises a cytokine IL-5 full-length polypeptide as a target recognition domain.

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

In some embodiments, the extracellular domain further comprises the proximal membrane end of CD28, that is, the full-length polypeptide derived from the above-mentioned IL-5 molecule is used as the target recognition structure and the proximal membrane end of CD28 is combined as the extracellular domain (extracellular segment).

(Transmembrane domain)

In some embodiments, the transmembrane domain comprises a transmembrane domain derived from a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

In some specific embodiments, the transmembrane domain comprises a transmembrane domain derived from CD28.

(Intracellular Signaling Domain)

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

In some embodiments, the primary intracellular signaling domain can comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences comprising ITAMs include, but are not limited to, those derived from CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12.

In the present disclosure, "ζ" or alternatively "ζ chain", "CD3-ζ", "TCR-ζ" or "CD3 zeta" is defined as the protein provided as GenBank Accession No. BAG36664.1, or the equivalent residues from a non-human species (e.g., mouse, rabbit, primate, mouse, rodent, monkey, ape, etc.), and "ζ stimulatory domain" or alternatively "CD3-ζ stimulatory domain" or "TCR-ζ stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the ζ chain that are sufficient to functionally transmit the initial signal necessary for T cell activation.

In the present disclosure, "costimulatory molecules" refer to cognate binding partners on T cells that specifically bind to costimulatory 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 that are 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).

The costimulatory intracellular signal transduction domain can be the intracellular part of a costimulatory molecule.Costimulatory molecules can be represented in the following protein families: TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein) and activated NK cell receptor. 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 a ligand specifically bound to CD83, etc.

In some specific embodiments, the CD28 intracellular segment and the CD3 zeta intracellular segment are combined as the intracellular signaling domain (intracellular region of signal transduction) of CAR.

In the present disclosure, the CD28 molecule is designed as an extracellular hinge segment (or proximal 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), and the chimeric antigen receptor mediated signal transduction level formed is stronger (compared to other designs) and the killing ability is more significant, and the target cell clearance efficiency is higher. In some specific embodiments of the present disclosure, the sequence of the CD28 molecule is shown in SEQ ID NO: 14. In other specific embodiments of the present disclosure, the sequence of the CD28 molecule is shown in SEQ ID NO: 18.

In the present disclosure, the intracellular segment of CD3zeta used in the intracellular signaling domain contains three ITAM motifs to maximize the signal level. In some specific embodiments of the present disclosure, the sequence of the CD3zeta molecule is shown in SEQ ID NO: 15. In other specific embodiments of the present disclosure, the sequence of CD3zeta is shown in SEQ ID NO: 19.

(IL-5 chimeric antigen receptor)

In some preferred embodiments of the present disclosure, an IL-5 chimeric antigen receptor is provided, which is a CAR molecule that can effectively recognize target cells and induce downstream activation signals. Immune cells/recombinant immune cells (such as T cells/recombinant T cells) modified by this molecule can kill target cells in vitro, while T cells modified by IL-5 chimeric antigen receptors are adoptively transferred into recipient mice and do not proliferate or kill target cells.

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

( _a1 ) the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16;

( _a2 ) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16, and that has or partially has the function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16;

( _a3 ) an amino acid sequence in which one or more amino acid residues are truncated, added, substituted, deleted or inserted into the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16, and which has or partially has the function of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16; or

( _a4 ) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16 under stringent conditions, and the amino acid sequence has or partially has the function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16, and the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

<Biological Materials>

In some aspects, the present disclosure provides a biomaterial, wherein the biomaterial comprises at least one of the following _b1 ) to _b3 ):

_b1 ) a polynucleotide encoding the chimeric antigen receptor as described above;

_b2 ) a vector containing the polynucleotide described in _b1 );

_b3 ) A cell containing the vector described in _b2 ).

In some embodiments, a polynucleotide encoding a chimeric antigen receptor of the present disclosure is provided.

The polynucleotides disclosed herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

The polynucleotide encoding the chimeric antigen receptor of the present disclosure includes: a coding sequence encoding only the chimeric antigen receptor; a coding sequence of the chimeric antigen receptor and various additional coding sequences; a coding sequence of the chimeric antigen receptor (and optional additional coding sequences) and non-coding sequences.

The term "polynucleotide encoding a chimeric antigen receptor" may refer to a polynucleotide encoding the chimeric antigen receptor or a polynucleotide further comprising additional coding and/or non-coding sequences.

The present disclosure also relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present disclosure particularly relates to polynucleotides that can hybridize to the polynucleotides described in the present disclosure under stringent conditions. The stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions, or very high stringent conditions.

In some embodiments, an expression vector is provided, comprising a polynucleotide of the present disclosure.

In some embodiments, a cell is provided, comprising an expression vector of the present disclosure.

In some specific embodiments, the cells are immune cells. The sources and types of the immune cells can be found in the detailed description of the related immune cells in the recombinant immune cell section below.

<Recombinant immune cells>

In some aspects of the present disclosure, the present disclosure provides a recombinant immune cell, wherein the recombinant immune cell comprises:

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

(ii) a gene regulatory system capable of reducing or eliminating the expression and/or function of the BCOR gene and the ZC3H12A gene in immune cells;

Wherein, the structure for adoptive cell therapy specifically binds to antigens derived from eosinophils.

(Immune Cells)

In some embodiments, the recombinant immune cell is an immune cell derived from a mammal.

The mammals include, but are not limited to, primates (eg, humans, monkeys), cows, sheep, goats, alpacas, horses, dogs, cats, rabbits, rats, mice, and the like.

In the present disclosure, there is no particular limitation in principle 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 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. In some more preferred embodiments, the T cells are CD8 ⁺ T cells.

In some specific embodiments, the recombinant immune cells are recombinant T cells.

The above-mentioned recombinant T cells do not contain BCOR gene and ZC3H12A gene, or the biological functions of BCOR gene products and ZC3H12A gene products of the recombinant T cells are inhibited.

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

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

As will be described in detail later, in the above-mentioned recombinant T cells, the knockout is to knock out the BCOR gene and ZC3H12A gene of the target T cells by the CRISPR-Cas9 method or other methods, or to inhibit the functions of the BCOR gene product and the ZC3H12A gene product by other methods.

In the above-mentioned recombinant T cells, the target sequence for targeting the BCOR gene when the BCOR gene in the target T cells is knocked out by the CRISPR-Cas9 method is SEQ ID NO: 4 or SEQ ID NO: 10; the target sequence for targeting the ZC3H12A gene when the ZC3H12A gene in the target T cells is knocked out by the CRISPR-Cas9 method is SEQ ID NO: 5 or SEQ ID NO: 11. Furthermore, the recombinant cells are vectors that carry the target sequence for targeting the BCOR gene when knocked out, the target sequence for targeting the ZC3H12A gene, and the expression of the IL-5CAR structure, and are introduced into the target T cells.

Further described in detail: 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. Exemplarily, the target CD8 T cells are derived from the spleen of Cas9 transgenic mice (from Jaxson Laboratory, Stock No: 026430) to obtain CD8 T cells.

(Gene Regulation System)

The recombinant immune cells contain a gene regulatory system capable of reducing or eliminating the expression and/or function of the BCOR gene and the ZC3H12A gene in the immune cells, so that the expression and/or function of the BCOR gene and the ZC3H12A gene in the recombinant immune cells are reduced or eliminated. Exemplarily, the recombinant T cells do not contain the BCOR gene and the ZC3H12A gene, or the biological functions of the BCOR gene product and the ZC3H12A gene product of the recombinant T cells are inhibited.

In the disclosure, exemplary information for the BCOR gene and the ZC3H12A gene can be found in Table 1 below.

Table 1 BCOR gene and ZC3H12A gene information

In the present disclosure, specifically, the human BCOR gene (Gene ID: 54880, updated on May 29, 2022, https://www.ncbi.nlm.nih.gov/gene/54880) and the mouse Bcor gene (Gene ID: 71458, updated on 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 on May 22, 2022, https://www.ncbi.nlm.nih.gov/gene/80149) and the mouse Zc3h12a gene (Gene ID: 230738, updated on May 22, 2022, https://www.ncbi.nlm.nih.gov/gene/230738) encode the protein ZC3H12A involved in mRNA degradation in cells. The above genes are all incorporated into the present disclosure by reference.

In the present disclosure, there is no particular limitation on the method used by the gene regulatory system to reduce or eliminate the expression and/or function of the BCOR gene and the ZC3H12A gene. For example, in some embodiments, the gene regulatory system can use gene knockout technology, gene silencing technology, inactivation mutation technology, PROTAC technology or small molecule inhibitors to treat the BCOR gene and the ZC3H12A gene in the recombinant immune cell.

In some embodiments, the gene regulatory system in the recombinant immune cells of the present 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, compared to unmodified or control immune cells (not having the gene regulatory system).

In some specific embodiments, the gene regulation system of the present disclosure uses gene knockout technology, gene silencing technology, or inactivation mutation technology or small molecule inhibitors to treat the BCOR gene and ZC3H12A gene in the recombinant immune cells.

In some embodiments, the gene knockout technology used includes CRISPR/Cas technology, artificial zinc finger nuclease (Zinc Finger Nucleases, ZFN) technology, transcription activator-like effector (TALE) technology or TALE-CRISPR/Cas technology.

In some embodiments, the gene regulation system comprises a nucleic acid molecule and an enzyme protein, wherein the nucleic acid molecule is a guide RNA (gRNA) molecule, and the enzyme protein is a Cas protein or a Cas ortholog.

In some embodiments, the enzyme protein is selected from Cas9, Cas12a, Cas12b, Cas13a, Cas13b, Cas13c, Cas13e or Cas13f protein or its direct homologs.

In some embodiments, the gene regulation system of the present disclosure comprises:

(i) a targeting domain sequence in a guide RNA (gRNA) targeting the BCOR gene is complexed with a first Cas endonuclease protein to form a first ribonucleoprotein (RNP) complex; and;

(ii) The targeting domain sequence in the ZC3H12A gene-targeting guide RNA (gRNA) is complexed with a second Cas endonuclease protein to form a second ribonucleoprotein (RNP) complex.

In some embodiments, the first ribonucleoprotein (RNP) complex and the second ribonucleoprotein (RNP) complex can be introduced into the immune cell simultaneously, sequentially or one after the other.

In some embodiments, in the gene regulation system of the present invention, the nucleic acid binding segment in the guide RNA (gRNA) targeting the BCOR gene binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a DNA sequence encoded by a BCOR gene derived from a subject (e.g., NCBI Gene ID: 54880 or NCBI Gene ID: 71458); the nucleic acid binding segment in the guide RNA (gRNA) targeting the ZC3H12A gene binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a DNA sequence encoded by a ZC3H12A gene derived from a subject (e.g., NCBI Gene ID: 80149 or NCBI Gene ID: 230738).

In some specific embodiments, in the gene regulation system of the present disclosure, the targeting domain in 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 in 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: NO:11 has a sequence with at least 85%, 90%, or 95% identity.

(Structure for adoptive cell therapy)

In some embodiments, the recombinant immune cells described herein include one or more structures for adoptive cell therapy.

In some embodiments, the corresponding structure used for adoptive cell therapy is a chimeric antigen receptor (CAR) structure, a T cell antigen receptor (TCR) structure, a receptor structure based on ligand receptor binding, or a 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 machinery mediate robust responses against solid tumors. Sci Transl Med. 2021Mar 24; 13(586):eabb5191.doi:10.1126/scitranslmed.abb5191.

In some embodiments, the antigen derived from eosinophils includes one or more of IL-5Rα, CRTh2, CCR3, Siglec-8. In some embodiments, the antigen derived from eosinophils includes IL-5Rα. In some more preferred embodiments, the antigen derived from eosinophils is IL-5Rα.

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 a chimeric antigen receptor provided above by the present disclosure.

(Biomolecules for the treatment of diseases)

In some embodiments, the recombinant immune cells provided by the present disclosure further include:

(iii) Biomolecules for the treatment of diseases.

In some specific embodiments, the biological molecules for treating diseases are selected from: cytokines, hormones, growth factors, coagulation factors, chemokines, co-stimulatory molecules, activation peptides, antibodies or their antigen-binding fragments, or mutants of the above. Compared with the above molecules in natural form, mutants will show different biological functions and have potential clinical effects for treating different diseases. Specifically, the biological molecules for treating diseases are 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 of the mutants of the above.

In some specific embodiments, the biological molecule for treating the disease is an IL-4 mutant, whose amino acid sequence is shown in SEQ ID NO: 7 or SEQ ID NO: 20, and which has the function of inhibiting IL-4 and IL-13 pathways.

In some embodiments, the recombinant immune cells of the present disclosure can be detected in the peripheral blood of the subject after 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, 40 years of administration to the subject. In some embodiments, the recombinant immune cells of the present disclosure are quasi-immortalized immune cells. Such immortalized recombinant immune cells are non-tumor cells.

In some embodiments, after administration to a 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 disclosed herein relative to the total amount of similar immune cells is not less than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

In other embodiments, after administration to a 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 disclosed herein relative to the total number of peripheral blood cells is selected from 1%-35%, 3-30%, or 3-20%; the specific numerical value may be any value within the above numerical range, including but not limited to 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, or 35%.

In other embodiments, compared with unmodified immune cells, the recombinant immune cells described in the present disclosure show increased or prolonged cell viability. In such embodiments, the result is that after a given time period, compared with unmodified immune cells, the number of recombinant immune cells of the present disclosure present increases. For example, in some embodiments, the recombinant immune cells described in the present disclosure maintain viability and last 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.

In some embodiments, the production of a disease treating biomolecule (e.g., IL23R, TNF or IL-5, GLP1, IL-4 mutant) by the recombinant immune cells described herein is increased by 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 times as compared to the production of the disease treating biomolecule observed in an unmodified immune cell population.

The recombinant immune cells provided by the present disclosure have increased immune cell stemness, inhibited immune cell exhaustion, promoted immune cell expansion, conferred immune cell memory, prolonged immune cell persistence, and increased immune cell self-renewal ability. As demonstrated in the examples, long-term protection can be achieved in an asthma mouse model without pretreatment.

<Method for preparing recombinant immune cells>

In some aspects, the present disclosure provides a method for preparing the above-mentioned recombinant immune cell, the method comprising:

(i) a step of introducing a construct for adoptive cell therapy into immune cells; and,

(ii) A step of introducing a gene regulatory system into immune cells.

(Steps for introducing gene regulation systems into immune cells)

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

In some embodiments, the gene knockout technology includes CRISPR/Cas technology, artificial zinc finger nucleases (Zinc Finger Nucleases, ZFN) technology, transcription activator-like effector (transcription activator-like effector, TALE) technology or TALE-CRISPR/Cas technology.

In some embodiments, the CRISPR/Cas technology is selected from CRISPR-Cas3, CRISPR-Cas9, CRISPR-Cas12, CRISPR-Cas13, CRISPR-CasX or CRISPR-IscB system. For example, the description of the CRISPR-CasX system is provided by Liu J.J. et al., Nature, 2019 or https://doi.org/10.1016/j.molcel.2022.02.002. For the description of the CRISPR-IscB system, see Han Altae-Tran. et al., Science 374, Vol 374, Issue 6563, 57-65 (2021). DOI: 10.1126/science.abj6856.

In some embodiments, 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 system.

In some embodiments, CRISPR/Cas technology uses a guide RNA (gRNA) and a Cas endonuclease targeting the BCOR gene, and a guide RNA (gRNA) and a Cas endonuclease targeting the ZC3H12A gene.

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

The present disclosure provides a guide RNA (gRNA) that directs a site-directed modification polypeptide to a specific target nucleic acid sequence. The gRNA comprises a nucleic acid targeting segment and a protein binding segment. The nucleic acid targeting segment of the gRNA comprises a nucleotide sequence that is complementary to a sequence in the target nucleic acid sequence. Therefore, the nucleic acid targeting segment 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 segment determines the position in the target nucleic acid to which the gRNA will bind. The nucleic acid targeting segment of the gRNA can be modified (e.g., by genetic engineering) to hybridize with any desired sequence in the target nucleic acid sequence.

The protein binding segment of the guide RNA interacts with the site-directed modification polypeptide (e.g., Cas protein) to form a complex. The guide RNA guides the bound polypeptide to a specific nucleotide sequence in the target nucleic acid through the above-mentioned nucleic acid targeting segment. The protein binding segment of the guide RNA comprises two nucleotide fragments, which are complementary to each other and form a double-stranded RNA duplex.

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

The specificity of the gRNA to the target locus is mediated by the sequence of the nucleic acid binding segment, which comprises about 20 nucleotides complementary to the target nucleic acid sequence within the target locus. In some embodiments, the length of the corresponding target nucleic acid sequence is about 20 nucleotides. In some embodiments, the nucleic acid binding segment 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 segment 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 segment 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.

In some embodiments, the target nucleic acid sequence in the target locus must be changed. 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 many examples of target nucleic acid sequences of gRNA in the specification and table provided herein. Any of these target nucleic acid sequences can be changed by moving the target nucleic acid sequence 5' or 3' in the target locus in a given gene. In some embodiments, the target nucleic acid sequence moves up to 100bp in the 5' or 3' in the target locus in a given gene. In other embodiments, the target nucleic acid sequence is moved at most 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 5' or 3' within the target locus within a given gene (e.g., the human or mouse BCOR gene and/or ZC3H12A gene described in Table 1).

In some embodiments, the nucleic acid binding segment in the guide RNA (gRNA) targeting the BCOR gene binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a DNA sequence encoded by a BCOR gene derived from a subject (e.g., NCBI Gene ID: 54880 or NCBI Gene ID: 71458); the nucleic acid binding segment in the guide RNA (gRNA) targeting the ZC3H12A gene binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a DNA sequence encoded by a ZC3H12A gene derived from a subject (e.g., NCBI Gene ID: 80149 or NCBI Gene ID: 230738).

In some embodiments, the nucleic acid binding segment in the guide RNA (gRNA) targeting the ZC3H12A gene binds to a target DNA sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to 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 gRNA molecule targeting ZC3H12A binds to a target DNA sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to one of the target DNA sequences shown in Tables 16 and 17 of WO2020163365A2.

In some embodiments, the targeting domain in the guide RNA (gRNA) targeting the BCOR gene comprises the sequence ACTGGGCAATACCGCAACAG (SEQ ID NO: 4) or a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 4; the targeting domain in the guide RNA (gRNA) targeting the BCOR gene comprises the sequence GCTGCCACAAGCACTCTAGG (SEQ ID NO: 10) or a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 10. column; the targeting domain of the guide RNA (gRNA) targeting the ZC3H12A gene comprises the sequence CTAGGGGAATTGGTGAAGCA (SEQ ID NO: 5) or a sequence that is at least 85%, 90%, 95% identical to 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 that is at least 85%, 90%, 95% identical to SEQ ID NO: 11.

(Step of introducing a structure for adoptive cell therapy into immune cells)

In some embodiments, in the step of introducing a structure for adoptive cell therapy into immune cells, a CAR structure or TCR structure with a target or other sequence of a corresponding structure for adoptive cell therapy is introduced into the immune cells. As mentioned above, the target can be an antigen derived from eosinophils.

In some embodiments, the antigen derived from eosinophils includes one or more of IL-5Rα, CRTh2, CCR3, Siglec-8. In some embodiments, the antigen derived from eosinophils includes IL-5Rα. In some more preferred embodiments, the antigen derived from eosinophils is IL-5Rα.

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 a chimeric antigen receptor provided above by the present disclosure.

(Step of introducing biomolecules for treating diseases into immune cells)

In some embodiments, the method for preparing recombinant immune cells provided by the present disclosure further comprises:

(iii) A step of introducing biomolecules for treating diseases into immune cells.

In some preferred embodiments, the biological molecule for treating 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, and one or more mutants thereof.

(Import method)

In the present disclosure, there are no particular limitations on the methods of introducing structures for adoptive cell therapy, introducing gene regulatory systems, and introducing biological molecules for treating diseases. For example, nucleic acids carrying structures capable of expressing adoptive cell therapy, one or more components of gene regulatory systems, and/or biological molecules for treating diseases can be introduced into immune cells using techniques known to those skilled in the art.

In some embodiments, the vector used is a viral vector, a virus-like vector or a non-viral vector. In some embodiments, the recombinant vector comprising a polynucleotide encoding a structure for adoptive cell therapy described herein, one or more components of a gene regulatory system (e.g., components of a gene regulatory system for reducing or eliminating the expression and/or function of BCOR and ZC3H12A genes in immune cells, such as sgRNA, Cas protein, etc.) and/or a biological molecule for treating a 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), etc. Suitable non-viral vectors are selected from transposons, lipid nanoparticles, liposomes, exosomes, attenuated bacteria, or virus-like particles.

In some embodiments, the polynucleotide sequences encoding the structures for adoptive cell therapy described herein, one or more components of the gene regulatory system, and/or the biomolecules for treating diseases are operably connected to control elements, such as transcriptional control elements, such as promoters. Transcriptional control elements can be functional in eukaryotic cells (e.g., mammalian cells) or prokaryotic cells (e.g., bacterial or archaeal cells). In some embodiments, the polynucleotide sequences encoding the structures for adoptive cell therapy described herein, one or more components of the gene regulatory system, and/or the biomolecules for treating diseases are operably connected to multiple control elements, which allow the polynucleotides 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 expression vectors.

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 repeats (LTR) from retroviruses, and mouse metallothionein-1. The selection of suitable vectors and promoters is entirely within the capabilities of those of ordinary skill in the art. The expression vector may also include a ribosome binding site and a transcription terminator for translation initiation. The expression vector may also include a suitable sequence for amplifying expression. The expression vector may also include a nucleotide sequence encoding a protein tag (e.g., 6xHis tag, hemagglutinin tag, green fluorescent protein, etc.) fused to the site-directed modified polypeptide to produce a chimeric polypeptide.

In some specific embodiments, exemplarily, the sgRNA expression vector used includes: the basic structure of vector-promoter 1-sgZc3h12a-promoter 2-tag-P2A-disease-treating biological molecule sequence, vector-promoter 1-sgBcor-promoter 2-tag-P2A-disease-treating biological molecule sequence or vector-promoter 1-sgBcor-promoter 2-sgZc3h12a-promoter 3-tag-P2A-disease-treating biological molecule sequence. The above "-" does not represent a limitation on a specific connection order, and should be understood as an expression vector containing related elements. The above-mentioned disease-treating biological molecule sequence includes one or more of the sequences of the structure for adoptive therapy in the recombinant immune cells described in the present disclosure or the sequences of biological molecules for treating diseases. Specifically, the sgRNA expression vector includes the basic structure 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, each promoter in the expression vector, such as promoter 1, promoter 2 and promoter 3, can be the same or different; the label is optionally present or absent; the biological molecule sequence for treating the disease is optionally present or absent. In other embodiments, sgBcor, sgZc3h12a, structures for adoptive therapy, biological molecules for treating diseases, etc. can also be constructed separately or in any number of combinations in a vector to form multiple expression vectors (such as viral vectors), which are packaged into corresponding viruses and co-transduced into immune cells.

In some embodiments, the present disclosure prepares a method for recombinant immune cells, including the step of introducing an expression vector into the recombinant immune cells. The method of introducing polynucleotides and recombinant expression vectors into host cells is known in the art, and any known method can be used to introduce the components of the gene regulation system into cells. Suitable methods include, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, 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 method, etc. In addition, cells can also be introduced into non-viral delivery vehicles, such as transposons, nanoparticles (e.g., lipid nanoparticles), liposomes, exosomes, attenuated bacteria or virus-like particles.

(Other steps)

In some embodiments, the method for preparing recombinant immune cells provided by the present disclosure further comprises the step of obtaining immune cells. In the present disclosure, there is no particular limitation in principle on the method for obtaining immune cells. Exemplarily, peripheral blood mononuclear cells can be isolated from the peripheral blood of the subject, and immune cells of a specific phenotype can be isolated by, for example, magnetic bead sorting or flow cytometry sorting techniques.

In some embodiments, the preparation method of the recombinant immune cells provided by the present disclosure also includes the step of recombinantly culturing immune cells. In the present disclosure, there is no particular limitation in principle for the method of culturing immune cells. In some embodiments, the recombinant immune cells can be implanted into the subject for amplification, and the recombinant immune cells amplified in vivo are obtained. The recombinant immune cells obtained after amplification from the first generation subject can be used for autologous treatment of the subject or for allogeneic treatment of other subjects. In some embodiments, the immune cells are autologous immune cells for the subject, or allogeneic immune cells.

<Composition>

In other aspects, the present disclosure provides a composition for treating a disease. In some embodiments, a "composition" refers to a preparation of a recombinant immune cell that is genetically regulated and/or modified, a chimeric antigen receptor that is provided by the present disclosure, and/or a biomaterial that is provided by the present disclosure, which can be administered or delivered to a subject or cell. A "therapeutic composition" or "pharmaceutical composition" (used interchangeably herein) is a composition comprising a recombinant immune cell that is genetically regulated and/or modified, a chimeric antigen receptor that is provided by the present disclosure, and/or a biomaterial that is provided by the present disclosure, which can be administered to a subject to treat a specific disease or condition.

In some embodiments, the composition for treating a disease comprises the recombinant immune cell described in any of the above embodiments. In some embodiments, the composition for treating a disease comprises the chimeric antigen receptor described in any of the above embodiments. In some embodiments, the composition for treating a disease comprises the biomaterial described in any of the above embodiments.

In some optional embodiments, the composition for treating a disease further comprises a pharmaceutically acceptable carrier.

<Methods for treating diseases or conditions>

In some embodiments, the present disclosure provides a method for treating a disease or condition in a subject in need thereof, comprising administering to the subject a recombinant immune cell as described in any of the above embodiments, a chimeric antigen receptor as described in any of the above embodiments, a biomaterial as described in any of the above embodiments, and/or administering a composition as described in any of the above embodiments.

In some embodiments, the present disclosure provides the recombinant immune cell 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 for use in treating and/or preventing a disease or condition in a subject.

In some embodiments, the disease or disorder comprises an inflammatory disease or allergic disease mediated by a type 2 immune response, or a disease in which eosinophils are effector cells.

In some specific embodiments, the inflammatory diseases or allergic diseases mediated by the type 2 immune response 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 disclosure.

In some specific embodiments, diseases in which eosinophils are (main) effector cells include: acute and chronic asthma, eosinophilia, nasal polyps caused by eosinophils, enteritis caused by eosinophils, eosinophilic dermatitis, chronic obstructive pulmonary disease, eosinophilic leukemia, etc.

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

<Pharmaceutical and other uses>

In some aspects, the present disclosure provides a use for preparing a medicament.

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

In some embodiments, the disease or disorder comprises an inflammatory disease or allergic disease mediated by a type 2 immune response, or a disease in which eosinophils are effector cells.

In other aspects, the present disclosure provides non-therapeutic uses of the recombinant immune cells described in any of the above embodiments.

In some embodiments, the non-therapeutic purpose includes using the recombinant immune cells for the preparation of a protein that can be encoded by DNA or for the preparation of a therapeutic composition. Preferably, the protein that can be encoded by DNA includes any one or more biological molecules for treating a disease described in any of the above embodiments.

<Application of delivery vector>

In another embodiment, the present disclosure provides a use of a recombinant immune cell as a carrier for stably delivering biological molecules for treating a disease.

In some embodiments, the recombinant immune cell is a recombinant immune cell as described in any one of the above embodiments. The biological molecule for treating a disease is selected from any one or more biological molecules for treating a disease as described in any one of the above embodiments.

In some embodiments, the biological molecules for treating diseases are selected from: cytokines, hormones, growth factors, coagulation factors, chemokines, co-stimulatory molecules, activation peptides, antibodies or their antigen-binding fragments, or mutants of the above. Specifically, the biological molecules for treating diseases are 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.

In order to make the purpose and technical solution of the present disclosure clearer, the embodiments of the present disclosure are described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure.

The reagents and instruments used in the examples are all commercially available conventional products. If no specific conditions are specified, conventional conditions or conditions recommended by the manufacturer are used.

Example 1. Preparation of recombinant IL-5CAR-T cells with knockout of Bcor and/or Zc3h12a

1. Construction of IL-5CAR expression vector

This embodiment designs and constructs a retrovirus-based IL-5CAR expression vector, namely pMSCV-EFS-Thy1.1-P2A-IL-5-CAR: wherein the pMSCV vector comes from Addgene#52107, and the IL-5CAR includes mouse IL-5 molecules, CD28 molecules (including the extracellular segment of the CD28 molecule, the membrane segment and the intracellular signal transduction region) and CD3zeta molecules. The above-constructed IL-5CAR molecule, the extracellular segment is a full-length IL-5 molecule of human or mouse origin, and the mouse-derived CD28 extracellular segment is used as the extracellular structure of the CAR molecule, and the intracellular segment is a mouse-derived CD28 and CD3zeta signal transduction domain, which is finally fused with the Thy1.1 tag for expression.

Among them, the amino acid sequence of IL-5CAR is (SEQ ID NO: 1):

The single underlined part is IL-5 (SEQ ID NO: 13); the double underlined part is the CD28 molecule (SEQ ID NO: 14); and the dotted underlined part is the CD3zeta molecule (SEQ ID NO: 15).

2. Construction of gene knockout IL-5CAR vector

In this example, based on the IL-5CAR expression vector constructed in 1 above, a retrovirus-based sgRNA expression vector was constructed, 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;

in:

Vector pMSCV-hU6-sgControl-EFS-Thy1.1-P2A-IL-5-CAR (SEQ ID NO: 2), in which positions 242-261 are random sequences SEQ ID NO: 3 that do not target any gene, serving as a control in which no gene is knocked out;

Vector pMSCV-hU6-sgBcor-EFS-Thy1.1-P2A-IL-5-CAR (the vector sequence is obtained by replacing the 242-261th position of SEQ ID NO: 2 with SEQ ID NO: 4, and keeping the other sequences unchanged. SEQ ID NO: 4 is the target sequence recognition region of sgBcor for knocking out Bcor, which is used to knock out Bcor;

Vector pMSCV-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR (the vector sequence is obtained by replacing positions 242-261 of SEQ ID NO: 2 with SEQ ID NO: 5, and keeping other sequences unchanged. SEQ ID NO: 5 is the target sequence recognition region of sgZc3h12a for knocking out Zc3h12a, and is used for knocking out Zc3h12a;

pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR (constructed on the basis of pMSCV-hU6-sgControl-EFS-Thy1.1-P2A-IL-5-CAR, by molecular cloning enzyme ligation method, hU6-sgBcor in the vector pMSCV-hU6-sgBcor-EFS-Thy1.1-P2A-IL-5-CAR and the vector pMSCV-hU6-sgZc3h12a-EFS- hU6-sgZc3h12a in Thy1.1-P2A-IL-5-CAR was amplified and spliced by PCR and then replaced into the position of hU6-sgControl), where positions 242-261 were the target sequence recognition region of mouse sgBcor for knocking out Bcor (SEQ ID NO:4), and positions 687-706 were the target sequence recognition region of sgZc3h12a for knocking out mouse Zc3h12a (SEQ ID NO:5) for knocking out mouse Bcor and mouse Zc3h12 at the same time. All vectors were obtained by whole gene synthesis.

The sgRNAs used above are shown in Table 2 below:

Table 2 Target sequence recognition region of sgRNA

3. Isolation and activation of naive CD8 T cells

Initial CD8 T cells were isolated from the spleen of Cas9 transgenic mice (from Jaxson Laboratory, #026430) by magnetic bead sorting, and the cells were inoculated at a density of 10 ⁶ cells/well into a 12-well cell culture dish coated with 1 μg/ml anti-CD3 antibody (CD3ε, BioXcell#BE0001-1), and 2 ml of RPMI1640 medium (containing 5% fetal bovine serum and interleukin-2) was added. At the same time, 1 μg/ml anti-CD28 antibody (BioXcell#BE0015-1) was added for in vitro activation, that is, the cells were cultured in a 5% carbon dioxide 37°C incubator and then infected with the virus.

4. Construction of IL-5CAR-T cells

1) Retrovirus preparation

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

2) Retroviral infection

1×10 ⁶ CD8 T cells activated by in vitro culture in the above 3 were added with 1 ml of the retroviral supernatant obtained in step 1) and mixed, and then centrifuged horizontally at room temperature for 2 hours at 2000g. Then, the cells were placed in a carbon dioxide incubator for 4 hours, replaced with 2 ml of fresh RPMI1640 medium (containing 5% fetal bovine serum and 2 ng/ml interleukin-2) and continued to be cultured (this time was recorded as the time after infection), and Thy1.1-positive cells (Thy1.1-biotin, BioLegend#202510) were sorted by flow cytometry to obtain IL-5 CAR-T cells.

5. Construction of IL-5 knockout CAR-T cells

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 specific genes. The other steps remain unchanged. Finally, we obtained IL-5CAR-T cells with both Bcor and Zc3h12a knocked out (expressed as sgBcor/Zc3h12a), named 5T _IF , where IF stands for Immortal -like and Functional , which means "immortal-like T cells" in Chinese. The other two types of cells were named sgBcor IL-5CAR-T cells, sgZc3h12a IL-5CAR-T cells and sgControl IL-5CAR-T cells.

6. Phenotypic identification of IL-5CAR-T cells

1) Detection of IL-5CAR molecular membrane level expression

Two days after the IL-5CAR retrovirus infected CD8 T cells in 4 above, flow cytometry was used to analyze the cells cultured in vitro to detect the Thy1.1 positive rate and IL-5 positive rate. The results are shown in B in Figure 1. The vast majority of cells carrying the Thy1.1 tag, that is, cells successfully transduced with the CAR vector, successfully expressed the IL-5 molecule.

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

On the third day after the above 3IL-5CAR retrovirus infected CD8 T cells, a co-incubation killing experiment was performed with MC-38 cells of the mouse tumor cell line expressing IL-5Rα and not expressing IL-5Rα, with the ratio of effector cells to target cells being 1:1, and anti-EGFR CAR-T cells were used as a control. The results, as shown in C in Figure 1, show that IL-5CAR-T cells can significantly kill target cells expressing the target, and have no killing effect on cells that do not express the target. Compared with EGFR CAR-T cells, IL-5CAR-T cells can kill target cells very significantly, showing the specificity of IL-5CAR-T cells. At the same time, we compared the killing ability of IL-5CAR-T cells at different ratios of effector cells and target cells. The results, as shown in D in Figure 1, show that at a ratio of 1:8, IL-5CAR-T cells still showed 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 have the ability to specifically kill target cells (see E and F in Figure 1).

7. IL-5CAR-T cells cannot proliferate or kill target eosinophils when infused back into the body without pretreatment

The cell reinfusion process is shown in A of Figure 2: CD8 T cells are isolated from the spleen and lymph nodes of Cas9 transgenic mice, and CD8 T cells are activated by CD3/CD28 (the method is the same as 3 of Example 1); the activated CD8 T cells are then infected with the retrovirus obtained by transfection with pMSCV-EFS-Thy1.1-P2A-IL-5-CAR to obtain IL-5CAR-T cells, and the above cells obtained after infection for 24 hours are respectively infused into C57bl/B6 mice (hereinafter referred to as B6 mice) through the tail vein. The specific infusion method is as follows:

6-8 week old B6 mice weighing 20-25 g were divided into 2 groups, namely PBS group (4 mice) and IL-5CAR-T group (4 mice).

PBS group: an equal volume of PBS was re-infused into the mice, and 200 μl of PBS was re-infused into each mouse in the PBS group through the tail.

IL-5CAR-T group: The IL-5CAR-T cells prepared by the example method were prepared into a cell suspension with PBS and transfused into each mouse through the tail. Each mouse was transfused with 4×10 ⁵ CAR-T cells.

On the 7th day after reinfusion, the ratio of reinfused IL-5CAR-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 carries a Thy1.1 tag). At the same time, the ratio of target cell eosinophils in the peripheral blood of each mouse was analyzed by flow cytometry using Siglec-F antibody (surface marker molecule of eosinophils).

As shown in Figure 2B and Figure 2D, no CAR-T cells were detected in the PBS group (indicated by "PBS" in the figure) as a control; and no amplified CAR-T cells were detected in the IL-5CAR group (indicated by "IL-5CAR" in the figure) to which IL-5CAR-T cells were infused. At the same time, as shown in Figure 2C and Figure 2D, both the PBS group and the IL-5CAR-T cell group detected the same number of eosinophils, and the statistical results showed that there was no significant difference between the two groups. The above results show that without any treatment of the recipient mice, IL-5CAR-T cells cannot amplify or kill target cells.

In order to more clearly understand the in vivo phenotype of IL-5CAR-T cells, we set up another experiment, that is, to obtain the same IL-5CAR-T cells in the same way as in the above example. The amount of reinfused cells was increased in a gradient, as follows:

6-8 week old B6 mice weighing 20-25 g were divided into 4 groups, namely PBS group (4 mice) and different doses of cells were further divided into 3 groups, each with 4 mice.

PBS group: an equal volume of PBS was re-infused into the mice, and 200 μl of PBS was re-infused into each mouse in the PBS group through the tail.

IL-5CAR-T group: IL-5CAR-T cells prepared by the example method were prepared into a cell suspension with PBS and transfused into each mouse in each group via tail injection. The doses of transfusion into each mouse were 1×10 ⁶ , 3×10 ⁶ , and 5×10 ⁶ cells, respectively.

On the 7th and 28th days after reinfusion, the ratio of reinfused IL-5CAR-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 carries a Thy1.1 label). At the same time, the ratio of target cell eosinophils in the peripheral blood of each mouse was analyzed by flow cytometry using Siglec-F antibody (surface marker molecule of eosinophils). On the 28th day after reinfusion, the mice were euthanized, the spleen and bone marrow were removed, and the ratio of IL-5CAR-T cells to total CD8 T cells and the ratio of target cell eosinophils in these two organs were analyzed by flow cytometry.

As shown in Figure 2E, Figure 2F and Figure 2G, no CAR-T cells were detected in the PBS group (indicated by "PBS" in the figure) as a control; and no amplified CAR-T cells were detected in the IL-5CAR group (indicated by "1×10 ⁶ , 3×10 ⁶ , 5×10 ⁶ " in the figure) with different doses of IL-5CAR-T cells. At the same time, both the PBS group and the different doses of IL-5CAR-T cell groups detected the same number of eosinophils, and the statistical results showed that there was no significant difference between the groups. The above results show that without any treatment of the recipient mice, increasing the number of IL-5CAR-T cells infused back cannot improve the proliferation and killing function of the cells. Similar results are shown in Figure 2H, Figure 2I and Figure 2J. In the main target cell organs and CAR-T cell aggregation organs, no CAR-T cells were detected, and the same number of eosinophils were detected, and the statistical results showed that there was no significant difference between the groups.

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

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

The cell reinfusion process is shown in A of Figure 2: CD8 T cells are isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3/CD28 for 24 hours, and activated CD8 T cells are obtained (the method is the same as 3 of Example 1); then the activated CD8 T cells are infected with the retrovirus 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 to obtain the corresponding gene knockout IL-5CAR-T cells, which are named, respectively. IL-5CAR-T cells, sgZc3h12a IL-5CAR-T cells, IL-5CAR-T cells with both Bcor and Zc3h12a knocked out (expressed as sgBcor/Zc3h12a), named 5T _IF , sgControl IL-5CAR-T cells. The above cells obtained after infection for 24 hours were respectively infused into C57bl/B6 mice (hereinafter referred to as B6 mice) through the tail vein, and the specific infusion method was as follows:

6-8 week old B6 mice weighing 20-25 g were divided into 2 groups, namely PBS group (4 mice), sgControl group (4 mice), sgZc3h12a group (4 mice), sgBcor group (4 mice), sgBcor/Zc3h12a group (5T _IF group, 4 mice)

PBS group: an equal volume of PBS was re-infused into the mice, and 200 μl of PBS was re-infused into each mouse in the PBS group through the tail.

sgControl group: sgControl IL-5CAR-T cells prepared by the method in step 5 of Example 1 were prepared into a cell suspension with PBS and transfused into each mouse through the tail. Each mouse was transfused with 4×10 ⁵ CAR-T cells.

sgBcor group: sgBcor IL-5CAR-T cells prepared by the method in step 5 of Example 1 were prepared into a cell suspension with PBS and transfused into each mouse through the tail. Each mouse was transfused with 4×10 ⁵ CAR-T cells.

sgZc3h12a group: sgZc3h12a IL-5CAR-T cells prepared by the method in step 5 of Example 1 were prepared into a cell suspension using PBS and transfused into each mouse through the tail. 4×10 ⁵ CAR-T cells were transfused into each mouse through the tail.

sgBcor/Zc3h12a group (5T _IF group): The sgBcor/Zc3h12a IL-5CAR-T (5T _IF ) cells prepared by the method in 5 of Example 1 were prepared into a cell suspension with PBS and infused back into each mouse through the tail. Each mouse was infused with 4×10 ⁵ CAR-T cells.

Two weeks after the mice were reinfused, the proportion of IL-5CAR-T cells in each group of mice in the peripheral blood of the mice was analyzed by flow cytometry to the total CD8T cells and the proportion of target cell eosinophils in the peripheral blood of each group. The results are shown in G in Figure 1 and H in Figure 1. Only in the sgBcor/Zc3h12a IL-5 group (5T _IF ) were significantly expanded IL-5CAR-T cells detected. At the same time, compared with the other groups, target cell eosinophils were almost undetectable in the peripheral blood of the sgBcor/Zc3h12a IL-5CAR-T group (5T _IF ). The above results show that only IL-5CAR-T cells with BCOR and ZC3h12a knocked out at the same time can significantly expand and significantly kill target cells, making the IL-5CAR molecule, which was originally only able to function in vitro, truly become a cell drug that can be used in vivo.

9. In vivo long-term phenotype identification of IL-5 knockout CAR-T cells

The cell reinfusion process is shown in A of Figure 2: CD8 T cells are isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3/CD28 for 24 hours, and activated CD8 T cells are obtained (the method is the same as 3 of Example 1); then the activated CD8 T cells are infected with the retrovirus obtained by transfecting pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR, and the IL-5CAR-T cells with Bcor and Zc3h12a (expressed as sgBcor/Zc3h12a) knocked out at the same time are named 5T _IF . The above cells obtained after 24 hours of infection were respectively infused into C57bl/B6 mice (hereinafter referred to as B6 mice) through the tail vein, and the specific infusion method is as follows:

6-8 week old B6 mice weighing 20-25 g were divided into 2 groups, namely PBS group (4 mice) and sgBcor/Zc3h12asgBcor/Zc3h12a group (5T _IF group, 4 mice).

PBS group: an equal volume of PBS was re-infused into the mice, and 200 μl of PBS was re-infused into each mouse in the PBS group through the tail.

sgBcor/Zc3h12a group (5T _IF group): The sgBcor/Zc3h12a IL-5CAR-T (5T _IF ) cells prepared by the method in 5 of Example 1 were prepared into a cell suspension with PBS and infused back into each mouse through the tail. Each mouse was infused with 4×10 ⁵ CAR-T cells.

The mice were counted for 24 weeks of reinfusion, and the proportion of IL-5CAR-T cells in each group of mice in the peripheral blood and the proportion of target cell eosinophils in the peripheral blood of each group were analyzed by flow cytometry. The results are shown in Figure 1 I and Figure 1 J. 5T _IF cells can be detected in the peripheral blood at different time points, and a peak appeared in the fourth week after reinfusion. Finally, the proportion of CAR-T cells in the peripheral blood tended to be stable. At the same time, compared with the PBS group, eosinophils were almost undetectable in the mice that had been reinfused with 5T _IF cells for a long time. The above results show that 5T _IF cells can not only proliferate significantly, but also persist in the mouse body for a long time, and can effectively kill target cells.

Example 2: 5T _IF recombinant cells expressing inhibitory IL-4 mutant proteins can effectively suppress type 2 immune responses

1. Construction of inhibitory IL-4 mutant protein vector

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

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

GFP amino acid sequence (SEQ ID NO: 8):

2. Construction of 5T _IF cells expressing inhibitory IL-4 mutant protein (i.e., 5T _IF 4 cells)

1) Retrovirus preparation

After 10 ⁶ Phoenix-Eco cells (ATCC #CRL-3214) were cultured for 24 hours, 20 μg of the gene knockout IL-5CAR expression vector pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR prepared in 2 of Example 1 or the pMSCV-EFS-GFP-P2A-IL-4Mutant (SEQ ID NO: 6) expression vector prepared in 1 of Example 2 and 60 μg of the packaging plasmid pCL-Eco (purchased from Addgene #12371) were co-transfected by the calcium phosphate precipitation method. The supernatant containing the packaged virus was harvested 48 hours after transfection, and the viral supernatant was filtered with a 0.45 μm filter membrane to remove dead cell impurities, and retroviral supernatants were obtained, i.e., a retrovirus with two genes knocked out and carrying IL-5CAR and a retrovirus expressing IL-4 mutant protein.

2) Retroviral infection

1×10 ⁶ CD8 T cells were activated and cultured in vitro for 36 hours in Example 1, and 1 ml of the two retroviral supernatants obtained in step 1) of Example 2 were added and mixed at 1:1, and then centrifuged at room temperature for 2 hours at 2000g. Then placed in a carbon dioxide incubator for 4 hours, replaced with 2 ml of fresh RPMI1640 medium (containing 5% fetal bovine serum and 2 ng/ml interleukin-2) and continued to be cultured (this time was recorded as the time after infection), and Thy1.1 and GFP double positive new cells (Thy1.1-biotin, BioLegend#202510) were sorted out by flow cytometry, that is, IL-5CAR-T cells expressing IL-4 mutant protein and knocking out two genes BCOR and ZC3h12a were obtained, named 5T _IF 4.

3. Detection and functional verification of inhibitory IL-4 mutant proteins

As shown in the method of A in Figure 4, two retroviruses are co-infected and flow-sorted to obtain double-positive cells, which are cultured and the culture supernatant is collected 48 hours after continued culture. At the same time, the viral supernatant of the CAR vector infected only with the pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR virus and the supernatant of cells not infected with the virus (as a control) are used. Centrifugation is performed for recovery, and the final centrifugation supernatant product is obtained by centrifugation. The content of IL-4 protein is detected by ELISA. The results are shown in B in Figure 3, and 5T _IF 4 cells can successfully secrete inhibitory mutant proteins.

The cell reinfusion process is shown in A of Figure 2: 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 (the method is the same as 3 of Example 1); 5T _IF cells were obtained by the method of 5 of Example 1; 5T _IF 4 cells were obtained by the method of 2 of Example 2. The above cells were respectively infused into C57bl/B6 mice (hereinafter referred to as B6 mice) through the tail vein, and the infusion method was as follows:

6-8 week old B6 mice weighing 20-25 g were divided into 2 groups, namely PBS group (4 mice), 5T _IF group (4 mice), 5T _IF 4 group (4 mice)

PBS group: an equal volume of PBS was re-infused into the mice, and 200 μl of PBS was re-infused into each mouse in the PBS group through the tail.

5T _IF group: The prepared 5T _IF cells were made into a cell suspension with PBS and infused back into each mouse through the tail. Each mouse was infused with 4×10 ⁵ CAR-T cells.

5T _IF 4 group: The prepared 5T _IF 4 cells were made into a cell suspension with PBS and infused back into each mouse through the tail. Each mouse was infused with 4×10 ⁵ CAR-T cells.

Five weeks after cell transfusion, mice were euthanized, blood was collected, cells were centrifuged, and supernatant serum was aspirated. The content of peripheral blood IL-4 mutant protein in the 5T _IF 4 group under the non-disease model was detected by ELISA. The results are shown in B in Figure 3. Compared with the recipient mice in the PBS group (control) and the 5T _IF group, the mice that were transfused with 5T _IF 4 cells had significantly increased IL-4 mutant protein levels.

The above results show that 5T _IF 4 cells can be used as a vector to express exogenous functional proteins.

4. Functional verification of inhibitory IL-4 mutant protein

The cell reinfusion process is shown in A of Figure 2: CD8 T cells are isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3/CD28 for 24 hours, and activated CD8 T cells are obtained (the method is the same as 3 of Example 1); 5T _IF cells are obtained by the method of 5 of Example 1; and 5T _IF 4 cells are obtained by the method of 2 of Example 2. The above cells are respectively infused into C57bl/B6 mice (hereinafter referred to as B6 mice) through the tail vein, and the infusion method is as follows:

6-8 week old B6 mice weighing 20-25 g were divided into 2 groups, namely PBS group (4 mice), 5T _IF group (4 mice), 5T _IF 4 group (4 mice)

PBS group: an equal volume of PBS was re-infused into the mice, and 200 μl of PBS was re-infused into each mouse in the PBS group through the tail.

5T _IF group: The prepared 5T _IF cells were made into a cell suspension with PBS and infused back into each mouse through the tail. Each mouse was infused with 4×10 ⁵ CAR-T cells.

5T _IF 4 group: The prepared 5T _IF 4 cells were made into a cell suspension with PBS and infused back into each mouse through the tail. Each mouse was infused with 4×10 ⁵ CAR-T cells.

Subsequently, as shown in the flowchart of C in Figure 3, mice that were infused with three groups of different substances were immunized, that is, a mixture of OVA protein and aluminum adjuvant was intraperitoneally injected to immunize the above-mentioned mice for weeks at intervals of 1 week. And 1 week after the secondary immunization, the mice were euthanized and serum and spleen were obtained respectively. The spleen was prepared with a single cell suspension, and the ratio of plasma cells and plasmablasts was detected by anti-B220 antibody and anti-CD138 antibody by flow cytometry. Cytokine IL-13 and total IgE in serum were detected by ELISA method.

As shown in D in Figure 3, 5T _IF 4 cells can significantly inhibit the level of IL-13 compared with 5T _IF cells. At the same time, as shown in E in Figure 3, both 5T _IF 4 cells and 5T _IF cells can significantly inhibit the level of IgE compared with the control group, and the inhibitory effect of 5T _IF 4 cells is more significant. As shown in F in Figure 3 and G in Figure 3, the analysis of B cell subsets in the spleen showed that 5T _IF 4 cells can effectively inhibit the generation of plasma cells compared with 5T _IF cells.

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

Example 3: 5T _IF 4 recombinant cells have the same long-term proliferation and killing target cell function

The cell reinfusion process is shown in A of Figure 2: CD8 T cells are isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3/CD28 for 24 hours to obtain activated CD8 T cells (the method is the same as 3 of Example 1); 5T _IF cells are obtained by the method of 5 of Example 1; and 5T _IF 4 cells are obtained by the method of 2 of Example 2.

A portion of the two obtained cells were cultured in vitro, and flow cytometry was used to detect the membrane efficiency and expression level of the CAR molecule. The results, as shown in Figure 4B and Figure 4C, showed that 5T _IF cells and 5T _IF 4 cells had no significant difference in the membrane efficiency of CAR molecules, and the levels of expression of CAR molecules in the two cells were consistent.

Another portion of the obtained cells were directly injected into C57bl/B6 mice (hereinafter referred to as B6 mice) through the tail vein. The specific injection method is as follows:

6-8 week old B6 mice weighing 20-25 g were divided into 2 groups, namely PBS group (4 mice), 5T _IF group (4 mice), 5T _IF 4 group (4 mice)

PBS group: an equal volume of PBS was re-infused into the mice, and 200 μl of PBS was re-infused into each mouse in the PBS group through the tail.

5T _IF group: The prepared 5T _IF cells were made into a cell suspension with PBS and infused back into each mouse through the tail. Each mouse was infused with 4×10 ⁵ CAR-T cells.

5T _IF 4 group: The prepared 5T _IF 4 cells were made into a cell suspension with PBS and infused back into each mouse through the tail. Each mouse was infused with 4×10 ⁵ CAR-T cells.

Two weeks after the mice were reinfused, the proportion of IL-5CAR-T cells in each group of mice in the peripheral blood to the total CD8T cells and the proportion of target cell eosinophils in the peripheral blood of each group were analyzed by flow cytometry. The results are shown in Figure 4D, Figure 4E and Figure 4F, showing that compared with 5T _IF , 5T _IF 4 also has the ability to proliferate and kill target cells for a long time. The above results show that the expression of exogenous proteins does not affect their own long-term killing function of target cells, and exogenous proteins can play independent functions in a bio-orthogonal manner.

Example 4: 5T _IF and 5T _IF 4 showed a curative effect in the acute asthma model induced by OVA antigen

1. Construction of OVA asthma model

The model was constructed by the method shown in A of Figure 5, as follows: 6-8 week old B6 mice weighing 20-25 g were selected, and each mouse was sensitized and immunized with 40 μg OVA protein mixed with aluminum adjuvant each time. On day 0 and day 7, the mice were immunized once by intraperitoneal injection, and then from day 16 to day 20, the mice were immunized by nasal drops at a dose of 40 μg per mouse. On day 21, 24 hours after the last immunization, the mice were euthanized, blood was collected to separate serum, lung lavage fluid was obtained through the airway, the lungs were separated, and single cell suspensions were obtained for subsequent flow cytometric analysis.

In another set of experiments, mice were perfused transcardially until the lungs turned white, and the lungs were finally isolated and fixed with paraformaldehyde for pathological sectioning and staining.

2. Cell transfusion and therapy

The cell reinfusion process is shown in A of Figure 2: CD8 T cells are isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3/CD28 for 24 hours to obtain activated CD8 T cells (the method is the same as 3 of Example 1); 5T _IF cells are obtained by the method of 5 of Example 1; and 5T _IF 4 cells are obtained by the method of 2 of Example 2.

The obtained cells were directly injected into C57bl/B6 mice (hereinafter referred to as B6 mice) through the tail vein at a specified time after OVA injection (as shown in A in FIG5 , day 9). The specific injection method is as follows:

The OVA-immunized mice were divided into 3 groups, namely PBS group (4 mice), 5T _IF group (4 mice), and 5T _IF 4 group (4 mice). PBS group: the 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 through the tail. 5T _IF group: the prepared 5T _IF cells were prepared into a cell suspension with PBS and re-infused into each mouse through the tail, and 4×10 ⁵ CAR-T cells were re-infused into each mouse through the tail. 5T _IF 4 group: the prepared 5T _IF 4 cells were prepared into a cell suspension with PBS and re-infused into each mouse through the tail, and 4×10 ⁵ CAR-T cells were re-infused into each mouse through the tail.

3. Efficacy testing

1) Flow cytometric analysis

The single cell suspension obtained from step 1 was stained with the following antibody groups: anti-CD45 antibody, anti-CD3e antibody, anti-B220 antibody, anti-CD11b antibody, anti-CD11c antibody, anti-Siglec-F antibody, and anti-Iy6G antibody, and finally detected by multicolor flow cytometer. Anti-Thy1.1 antibody and anti-CD8 antibody were used to detect CAR-T cells infiltrating the lungs. Subsequently, the quantitative cells were stained with anti-CD45 antibody and counted by flow cytometer.

The results are shown in Figure 5B and Figure 5C . 5T _IF and 5T _IF 4 cells can significantly infiltrate into the lungs, and there is no significant difference in their absolute numbers.

The analysis results of other immune cells showed that, as shown in Figure 5G and Figure 5H, 5T _IF and 5T _IF 4 cell therapy can significantly inhibit the infiltration of total blood cells in lung lavage fluid and lungs. Among them, both significantly inhibited the infiltration content of eosinophils, the main pathogenic cells infiltrating in lavage fluid and lungs, but the two did not show significant differences in inhibiting cell infiltration. The above results show that both cell therapies can almost eliminate eosinophils infiltrating the lungs.

2)ELISA

The cell-free supernatant of the lung lavage fluid obtained in step 1 was transferred into a clean EP tube, and the IL-13 content therein was detected using an ELISA kit. The serum obtained in step 1 was transferred into a clean EP tube, and the total IgE content therein was detected using an ELISA kit.

The results, as shown in Figure 5E and Figure 5F, show that both 5T _IF and 5T _IF 4 can significantly inhibit the production of IL-13, and the inhibitory effect of 5T _IF 4 is more significant. Similarly, both 5T _IF and 5T _IF 4 can significantly inhibit the production of IgE, and 5T _IF 4 has a more significant inhibitory effect in inhibiting the production of IgE. In summary, 5T _IF 4 shows better efficacy in inhibiting the production of cytokines and antibodies.

3) Pathological staining

The lungs fixed overnight were embedded in paraffin and then sliced and mounted; the prepared pathological sections were stained with HE and photographed under a high-power microscope. The pathological tissues were scored using a 1-4 grade pathological scoring method.

The pathological analysis results (D) in Figure 5 indicate that since both are very effective in killing target cells and inhibiting the infiltration of immune cells, the pathological staining results also show that 5T _IF and 5T _IF 4 can significantly inhibit the infiltration and distribution of immune cells in the lungs, and there is no significant difference between the two.

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 and alleviating the inflammatory response of pathological tissues. Furthermore, 5T _IF 4 cells are more effective in inhibiting cytokines and antibodies.

Example 5: 5T _IF and 5T _IF 4 showed a curative effect in a chronic asthma model induced by OVA antigen and repeatedly exposed to antigen

1. Construction of OVA asthma model

The model was constructed by the method shown in A of Figure 6, as follows: 6-8 week old B6 mice weighing 20-25g were selected, and each mouse was sensitized and immunized with 40μg OVA protein mixed with aluminum adjuvant each time. On day 0 and day 10, the mice were immunized once by intraperitoneal injection, and then from day 12 onwards, the mice were immunized with antigen exposure twice a week by nasal drops at a dose of 40μg per mouse, and the immunized mice were boosted with intraperitoneal OVA every month. From day 176 to day 179, the mice were immunized with terminal antigen exposure by nasal drops at a dose of 40μg per mouse. On day 180, 24 hours after the last immunization, the mice were euthanized, blood was collected to separate serum, lung lavage fluid was obtained through the airway, the lungs were separated, and single cell suspensions were obtained for subsequent flow cytometric analysis.

In another set of experiments, mice were perfused transcardially until the lungs turned white, and the lungs were finally isolated and fixed with paraformaldehyde for pathological sectioning and staining.

2. Cell transfusion and therapy

The cell reinfusion process is shown in A of Figure 2: CD8 T cells are isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3/CD28 for 24 hours to obtain activated CD8 T cells (the method is the same as 3 of Example 1); 5T _IF cells are obtained by the method of 5 of Example 1; and 5T _IF 4 cells are obtained by the method of 2 of Example 2.

The obtained cells were directly injected into C57bl/B6 mice (hereinafter referred to as B6 mice) through the tail vein at a specified time after OVA injection (as shown in A in FIG6 , day 12). The specific injection method is as follows:

The OVA-immunized mice were divided into 3 groups, namely PBS group (4 mice), 5T _IF group (4 mice), and 5T _IF 4 group (4 mice). PBS group: the 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 through the tail. 5T _IF group: the prepared 5T _IF cells were prepared into a cell suspension with PBS and re-infused into each mouse through the tail, and 4×10 ⁵ CAR-T cells were re-infused into each mouse through the tail. 5T _IF 4 group: the prepared 5T _IF 4 cells were prepared into a cell suspension with PBS and re-infused into each mouse through the tail, and 4×10 ⁵ CAR-T cells were re-infused into each mouse through the tail.

3. Efficacy testing

1) Flow cytometric analysis

The single cell suspension obtained from step 1 was stained with the following antibody groups: anti-CD45 antibody, anti-CD3e antibody, anti-B220 antibody, anti-CD11b antibody, anti-CD11c antibody, anti-Siglec-F antibody, and anti-Iy6G antibody, and finally detected by multicolor flow cytometer. Anti-Thy1.1 antibody and anti-CD8 antibody were used to detect CAR-T cells infiltrating the lungs. Subsequently, the quantitative cells were stained with anti-CD45 antibody and counted by flow cytometer.

As shown in Figure 6B and Figure 6C, 5T _IF and 5T _IF 4 cells can significantly infiltrate into the lungs, and there is no significant difference in their absolute number. The total number of cells in both cases has a significant downward trend compared with Example 4, indicating that the response of CAR-T is gradually slowing down.

The analysis results of other immune cells showed that, as shown in G and H in Figure 6, 5T _IF and 5T _IF 4 cell therapy can significantly inhibit the infiltration of total blood cells in lung lavage fluid and lungs. Among them, both significantly inhibited the infiltration content of eosinophils, the main pathogenic cells infiltrating in lavage fluid and lungs, but there was no significant difference in the inhibitory effect between the two. And both significantly inhibited the infiltration of T cells and B cells in the lungs. In summary, the results show that both cell therapies can almost eliminate eosinophils infiltrating the lungs.

2)ELISA

The cell-free supernatant of the lung lavage fluid obtained in step 1 was transferred into a clean EP tube, and the IL-13 content therein was detected using an ELISA kit. The serum obtained in step 1 was transferred into a clean EP tube, and the total IgE content therein was detected using an ELISA kit.

The results are shown in Figure 6E and F. 5T _IF 4 can significantly inhibit the production of IL-13. At the same time, 5T _IF can significantly inhibit the production of IgE. In summary, 5T _IF 4 shows better efficacy in inhibiting the production of cytokines and antibodies.

3) Pathological staining

The lungs fixed overnight were embedded in paraffin and then sliced and mounted; the prepared pathological sections were stained with HE and PAS, and the slices were photographed with a high-power microscope. The pathological tissues were scored using the 1-4 grade pathological scoring method.

The pathological analysis results in Figure 6D show that since both of them are very effective in killing target cells and inhibiting the infiltration of immune cells, the pathological staining results also show that 5T _IF and 5T _IF 4 can significantly inhibit the infiltration and distribution of immune cells in the lungs, and there is no significant difference between the two. The results of PAS staining clearly show that 5T _IF 4 significantly inhibits the increase and proliferation of PAS-positive goblet cells compared to 5T _IF .

Based on the above results, 5T _IF and 5T _IF 4 cell therapy can significantly remove pathogenic eosinophils, while inhibiting the infiltration of more immune cells and alleviating the inflammatory response of pathological tissues. Furthermore, 5T _IF 4 cells are more effective in inhibiting cytokines and antibodies. At the same time, 5T _IF 4 cells have a more obvious effect on alleviating pathological changes in the lungs.

Example 6: 5T _IF and 5T _IF 4 can effectively prevent IL-33-induced asthma

1. IL-33 respiratory sensitization model

The model was constructed by the method shown in A of Figure 7, as follows: 6-8 week old B6 genetic background mice weighing 20-25 g were selected and CAR-T cells were transfused into them. In the 5th week after transfusion, the mice were sensitized with IL-33, that is, from day 0 to day 3, the mice were exposed to nasal drops once each. On day 4, 24 hours after the last nasal drop immunization, the mice were euthanized, lung lavage fluid was obtained through the airway, the lungs were separated and single cell suspensions were obtained for subsequent flow cytometric analysis.

In another set of experiments, mice were perfused transcardially until the lungs turned white, and the lungs were finally isolated and fixed with paraformaldehyde for pathological sectioning and staining.

2. Cell transfusion and therapy

The cell reinfusion process is shown in A of Figure 2: CD8 T cells are isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3/CD28 for 24 hours to obtain activated CD8 T cells (the method is the same as 3 of Example 1); 5T _IF cells are obtained by the method of 5 of Example 1; and 5T _IF 4 cells are obtained by the method of 2 of Example 2.

The obtained cells were injected into B6 mice at the designated time (as shown in A in FIG7 ) in the following manner:

The mice were divided into 3 groups, namely PBS group (4 mice), 5T _IF group (4 mice), and 5T _IF 4 group (4 mice). PBS group: the 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 through the tail. 5T _IF group: the prepared 5T _IF cells were prepared into a cell suspension with PBS and re-infused into each mouse through the tail, and 4×10 ⁵ CAR-T cells were re-infused into each mouse through the tail. 5T _IF 4 group: the prepared 5T _IF 4 cells were prepared into a cell suspension with PBS and re-infused into each mouse through the tail, and 4×10 ⁵ CAR-T cells were re-infused into each mouse through the tail.

3. Prevention effect detection

1) Flow cytometric analysis

The single cell suspension obtained from step 1 was stained with the following antibody groups: anti-CD45 antibody, anti-CD3e antibody, anti-B220 antibody, anti-CD11b antibody, anti-CD11c antibody, anti-Siglec-F antibody, and anti-Iy6G antibody, and finally detected by multicolor flow cytometer. Anti-Thy1.1 antibody and anti-CD8 antibody were used to detect CAR-T cells infiltrating the lungs. Subsequently, the quantitative cells were stained with anti-CD45 antibody and counted by flow cytometer.

As shown in Figure 7B and Figure 7C, 5T _IF and 5T _IF 4 cells can significantly infiltrate into the lungs, indicating that local cytokines can also induce a strong CAR-T immune response.

The analysis results of other immune cells showed that, as shown in Figure 7F and Figure 7G, 5T _IF and 5T _IF 4 cell therapy can significantly inhibit the infiltration of total blood cells in lung lavage fluid and lungs. Among them, both significantly inhibited the infiltration content of eosinophils, the main pathogenic cells infiltrating in lavage fluid and lungs, but the two showed no significant difference in the inhibitory effect. And both significantly inhibited the infiltration of T cells and B cells in the lungs. In summary, the results show that both cell therapies can almost eliminate eosinophils infiltrating the lungs.

2)ELISA

The cell-free supernatant of the lung lavage fluid obtained in step 1 was transferred into a clean EP tube; at the same time, blood was collected from the mice, and the serum was separated and the IL-13 content therein was detected using an ELISA kit.

The results, as shown in Figure 7E, showed that 5T _IF 4 had a significant inhibitory effect on the production of IL-13.

3) Pathological staining

The lungs fixed overnight were embedded in paraffin and then sliced and mounted; the prepared pathological sections were stained with HE and photographed under a high-power microscope. The pathological tissues were scored using a 1-4 grade pathological scoring method.

D in the pathological analysis result 7 shows that since both are very significant in killing target cells and inhibiting the infiltration of immune cells, the pathological staining results also show that 5T _IF and 5T _IF 4 can significantly inhibit the infiltration and distribution of immune cells in the lungs, and there is no significant difference between the two.

Based on the above results, the reinfused 5T _IF and 5T _IF 4 cells can significantly remove pathogenic eosinophils, while inhibiting the infiltration of more immune cells and preventing the occurrence of inflammatory responses in pathological tissues. Furthermore, 5T _IF 4 cells are more effective in inhibiting cytokines. Mice that were reinfused with 5T _IF and 5T _IF 4 cells reshaped their own peripheral tolerance environment, including the tolerance environment of local organs, that is, they had the effect of tolerating and inhibiting inflammation to the stimulation of strong sensitizing cytokines.

Example 7: 5T _IF and 5T _IF 4 can effectively prevent HDM-induced asthma

1. HDM respiratory sensitization exposure model

The model was constructed by the method shown in A of Figure 8, as follows: 6-8 weeks old B6 genetic background mice weighing 20-25g were selected and CAR-T cells were transfused into them. At week 10 after transfusion, the mice were sensitized with house dust mites (HDM), that is, from day 7 to day 11, the mice were exposed to HDM by intranasal drops once each. On day 14, 72 hours after the last intranasal immunization, the mice were euthanized, lung lavage fluid was obtained through the airway, the lungs were separated and single cell suspensions were obtained for subsequent flow cytometry analysis.

In another set of experiments, mice were perfused transcardially until the lungs turned white, and the lungs were finally isolated and fixed with paraformaldehyde for pathological sectioning and staining.

2. Cell transfusion and therapy

The cell reinfusion process is shown in A of Figure 2: CD8 T cells are isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3/CD28 for 24 hours to obtain activated CD8 T cells (the method is the same as 3 of Example 1); 5T _IF cells are obtained by the method of 5 of Example 1; and 5T _IF 4 cells are obtained by the method of 2 of Example 2.

The obtained cells were directly injected into C57bl/B6 mice (hereinafter referred to as B6 mice) through the tail vein at the designated time (as shown in A in FIG8 ). The specific injection method is as follows:

The mice were divided into 3 groups, namely PBS group (4 mice), 5T _IF group (4 mice), and 5T _IF 4 group (4 mice). PBS group: the 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 through the tail. 5T _IF group: the prepared 5T _IF cells were prepared into a cell suspension with PBS and re-infused into each mouse through the tail, and 4×10 ⁵ CAR-T cells were re-infused into each mouse through the tail. 5T _IF 4 group: the prepared 5T _IF 4 cells were prepared into a cell suspension with PBS and re-infused into each mouse through the tail, and 4×10 ⁵ CAR-T cells were re-infused into each mouse through the tail.

3. Prevention effect detection

1) Flow cytometric analysis

The single cell suspension obtained from step 1 was stained with the following antibody groups: anti-CD45 antibody, anti-CD3e antibody, anti-B220 antibody, anti-CD11b antibody, anti-CD11c antibody, anti-Siglec-F antibody, and anti-Iy6G antibody, and finally detected by multicolor flow cytometer. Anti-Thy1.1 antibody and anti-CD8 antibody were used to detect CAR-T cells infiltrating the lungs. Subsequently, the quantitative cells were stained with anti-CD45 antibody and counted by flow cytometer.

As shown in Figure 8 B and Figure 8 C, 5T _IF and 5T _IF 4 cells can significantly infiltrate into the lungs, and there is no significant difference in their absolute number. And the total number of cells of the two is significantly reduced compared with the number under immunization in the previous embodiments, indicating that 5T _IF and 5T _IF 4 cells prevent local inflammatory response, and the corresponding CAR-T immune response also tends to decrease, but there is still significant infiltration.

The analysis results of other immune cells showed that, as shown in Figure 8F and Figure 8G, 5T _IF and 5T _IF 4 cell therapies can significantly inhibit the infiltration of total blood cells in lung lavage fluid and lungs. Among them, both significantly inhibited the infiltration content of eosinophils, the main pathogenic cells infiltrating in lavage fluid and lungs. Although the two did not show significant statistical differences in the inhibitory effect, 5T _IF 4 showed a more obvious inhibitory trend. And both significantly inhibited the infiltration of T cells and B cells in the lungs. The above results show that both cell therapies can almost eliminate eosinophils infiltrating the lungs.

2)ELISA

The cell-free supernatant of the lung lavage fluid obtained in step 1 was transferred into a clean EP tube; at the same time, blood was collected from the mice, and the serum was separated and the IL-13 content therein was detected using an ELISA kit.

The results, as shown in Figure 8E, showed that both 5T _IF and 5T _IF 4 cells could significantly inhibit the production of IL-13, and the inhibitory effect of 5T _IF was more significant.

3) Pathological staining

The lungs fixed overnight were embedded in paraffin and then sliced and mounted; the prepared pathological sections were stained with HE and photographed under a high-power microscope. The pathological tissues were scored using a 1-4 grade pathological scoring method.

The pathological analysis results (D) in Figure 8 indicate that since both cells are very effective in killing target cells and inhibiting the infiltration of immune cells, the pathological staining results also show that both 5T _IF and 5T _IF 4 cells can significantly inhibit the infiltration and distribution of immune cells in the lungs, and there is no significant difference between the two.

Based on the above results, the reinfused 5T _IF and 5T _IF 4 cells can significantly remove pathogenic eosinophils, while inhibiting the infiltration of more immune cells and preventing the occurrence of inflammatory responses in pathological tissues. Furthermore, 5T _IF 4 cells are more effective in inhibiting cytokines. Mice that were reinfused with 5T _IF and 5T _IF 4 cells reshaped their own peripheral tolerance environment, including the tolerance environment of local organs, that is, they had the effect of tolerating and inhibiting inflammation to the stimulation of strong sensitizing cytokines.

Example 8: Human h5T _IF4 cells can continuously kill target cells in NSG mice

1. Construction of human IL-5CAR, human gene-edited IL-5CAR vector and human IL-4 mutant protein vector

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

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

The sgRNAs used above are shown in Table 3 below:

Table 3 Target sequence recognition region of sgRNA

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

2. Isolation (recovery), activation and lentiviral transduction of human PBMC

1) Isolation and recovery of human PBMC: Human PBMC were separated by referring to Ficoll lymphocyte separation solution, and peripheral blood samples were diluted with PBS at a ratio of 1:1; lymphocyte separation solution was added at a ratio of 2:1; gradient centrifugation was performed at 800g for 25 minutes; after centrifugation, the middle layer was transferred to a new centrifuge tube, and PBS was added and washed twice; resuspended in PBS, counted, centrifuged, and resuspended in 100% serum and frozen at -80°C. The frozen human PBMC were rapidly revived at 37°C, and the cells were resuspended in human T cell culture medium and revived in a carbon dioxide incubator at 37°C for 6 to 12 hours.

2) Lentivirus packaging and concentration: After 10 ⁶ Lenti-X cells (ATCC#CRL-3214) were cultured for 20 hours, 30 μg of the expression vector prepared in 1 above and 20 μg of packaged psPAX2 (Addgene#12260) and 10 μg of packaged pMD2.G (Addgene#12259) were co-transfected by calcium phosphate precipitation method. The supernatant containing packaged virus, i.e., retrovirus supernatant, was harvested 48 hours after transfection. The obtained supernatant was concentrated by ultracentrifugation.

3) Activation and transduction of human T cells: Add fully recovered human PBMCs to cell culture plates coated with anti-CD3 antibodies and Retronectin and activate for 24 to 36 hours; after activation, add concentrated virus and infect for 24 to 36 hours; perform routine culture at a later stage.

3. Construction of h5T _IF 4 cells by electroporation and cell adoptive transfer

1) Electroporation of human T cells: The cultured human T cells were washed three times with PBS; 2 μl of Cas9 mRNA (Aibixin) was added to each well at a concentration of 2.5 to 4×10 ⁶ cells for electroporation reaction, mixed and allowed to stand for 2 minutes; electroporation was performed in a Lonza electroporator using the EO-115 program; the electroporated cells were allowed to stand in a carbon dioxide incubator for recovery, and then transferred to a 48-well plate for continued culture, as shown in FIG10A .

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

4. In vitro functional characterization of human IL-5CAR-T cells and h5T _IF 4 cells

1) Identification of the killing ability of human IL-5CAR-T cells in vitro: First, the expression of CAR molecules in human IL-5CAR-T cells was analyzed by FACS, and the results are shown in Figure 9 B. And through a 24-hour co-incubation experiment with 143B tumor cells expressing IL-5Ra (human osteosarcoma cells, from ATCC), the killing level was detected. The results show that human IL-5CAR-T cells can effectively kill target cells as shown in Figure 9 C. In addition, by changing the ratio of CAR-T cells and target cells, the killing level of CAR-T cells at different E:T ratios was detected, which is shown as D in the statistical results Figure 9.

2) Virus co-infection system and identification of expression level of IL-4 mutant protein: Two lentiviruses were constructed by expression vectors, such as E in FIG9 ; two viruses were added to the activated human T cell culture medium at the same time by co-infection, and the co-infection was detected by FACS at 36 or 48 hours after static infection, and the results are shown in F in FIG9 . The cell culture supernatant was collected, and the expression level of IL-4 was detected by ELISA kit, and the statistical results are shown in G in FIG9 .

3) Detection of gene editing level: Before adoptive transplantation into NSG, collect some cells, extract DNA, amplify the specific sites of gene editing by PCR, purify and recover the products, and sequence them. Detect the gene editing situation, and the representative gene result graph is shown in Figure 10H

5. Verification of h5T _IF 4 cell function in vivo

1) In vivo expansion and killing ability detection of h5T _IF 4 cells: In the fourth week of adoptive transplantation, blood was collected and FACS was used to detect the proportion of CAR-T cells and eosinophils in the peripheral blood. The results showed that compared with non-electroporated IL-5CAR-T cells, h5T _IF 4 cells could significantly expand and kill target cells. The results are shown in B, C and D of Figure 10.

2) Kinetic analysis of the expansion and killing ability of h5T _IF 4 cells: At the 2nd, 4th and 8th week after adoptive transplantation, blood was collected and the proportion of CAR-T cells and eosinophils in the peripheral blood was detected by FACS, and statistical graphs of dynamic changes were drawn, as shown in Figure 10 E and F. The results showed that h5T _IF 4 cells were able to kill target cells starting from the 2nd week after transplantation, and continued until the experimental endpoint of the 8th week. At the same time, the results showed that the proportion of CAR-T cells reached a peak in the 4th week after transplantation and lasted for 8 weeks. At the end of the 8th week, we euthanized the mice, extracted serum samples, and used the ELSIA method to detect the level of IL-4 released by h5T _IF 4 cells. The results are shown in Figure 10 G.

At the same time, this example also constructs a fully humanized IL-5CAR, whose sequence is (SEQ ID NO: 16):

The single underlined part is IL-5 (SEQ ID NO: 17); the double underlined part is the CD28 molecule (SEQ ID NO: 18); and the dotted underlined part is the CD3zeta molecule (SEQ ID NO: 19).

At the same time, this embodiment also constructed a fully humanized IL-4 mutant protein, whose sequence is (SEQ ID NO: 20): HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGA TAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMDEKDSKCSS*

And placed them on the same expression vector, namely pHAGE-SFFV-human IL-4mutant-P2A-human IL-5CAR, whose sequence is SEQ ID NO:21.

The homology between SEQ ID NO:16 and SEQ ID NO:1 as well as SEQ ID NO:20 and SEQ ID NO:7 is highly conserved, and it is predicted that they will have the same expected therapeutic effects.

Vector sequence

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

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

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

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

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

Claims (14)

A recombinant immune cell, wherein the recombinant immune cell comprises: (i) one or more structures for adoptive cell therapy; (ii) a gene regulatory system capable of reducing or eliminating the expression and/or function of the BCOR gene and the ZC3H12A gene in immune cells; Wherein, the structure for adoptive cell therapy specifically binds to antigens derived from eosinophils. The recombinant immune cell according to claim 1, wherein the antigen derived from eosinophils comprises one or more of IL-5Rα, CRTh2, CCR3, and Siglec-8; Preferably, the eosinophil-derived antigen is IL-5Rα. The recombinant immune cell according to claim 1 or 2, wherein the structure used for cell adoptive therapy is selected from one or more of a chimeric antigen receptor, a T cell antigen receptor, a receptor based on ligand receptor binding, and a synthetic T cell receptor and antigen receptor; Preferably, the structure for adoptive cell therapy is a chimeric antigen receptor, which comprises: (a) a polypeptide derived from IL-5; (b) a polypeptide derived from CD28; and, (c) A polypeptide derived from CD3zeta. The recombinant immune cell according to claim 3, wherein 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, The amino acid sequence of the polypeptide derived from CD28 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. The recombinant immune cell according to claim 3 or 4, wherein the chimeric antigen receptor comprises one or more of the following sequences: ( _a1 ) the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16; ( _a2 ) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:16, and that has or partially has the function of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:16; ( _a3 ) an amino acid sequence in which one or more amino acid residues are truncated, added, substituted, deleted or inserted into the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16, and which has or partially has the function of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16; or ( _a4 ) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16 under stringent conditions, and the amino acid sequence has or partially has the function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16, and the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions. The recombinant immune cell according to any one of claims 1 to 5, wherein the recombinant immune cell further comprises: (iii) biomolecules for the treatment of diseases; Optionally, the biological molecule for treating a disease is selected from the group consisting of: cytokines, hormones, growth factors, coagulation factors, chemokines, co-stimulatory molecules, activation peptides, antibodies or antigen-binding fragments thereof, or mutants thereof; Preferably, the biological molecule for treating a disease is selected from one or more of 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; More preferably, the biological molecule for treating the disease is an IL-4 mutant, whose amino acid sequence is shown in SEQ ID NO: 7 or SEQ ID NO: 20. The recombinant immune cell according to any one of claims 1 to 6, wherein the immune cell is an immune cell derived from a mammal; Optionally, the immune cells are selected from one or more of T cells, B cells, NK cells, mast cells, and tumor infiltrating lymphocytes; Preferably, the immune cells are selected from T cells or NK cells; 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. The recombinant immune cell according to any one of claims 1 to 7, wherein the gene regulation system uses gene knockout technology, gene silencing technology, inactivation mutation technology, PROTAC technology or small molecule inhibitors to process the BCOR gene and ZC3H12A gene in the recombinant immune cell. A chimeric antigen receptor comprising: (a) a polypeptide derived from IL-5; (b) a polypeptide derived from CD28; and, (c) a polypeptide derived from CD3zeta; Preferably, the chimeric antigen receptor comprises one or more of the following sequences: ( _a1 ) the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16; ( _a2 ) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16, and that has or partially has the function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16; ( _a3 ) an amino acid sequence in which one or more amino acid residues are truncated, added, substituted, deleted or inserted into the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16, and which has or partially has the function of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16; or ( _a4 ) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16 under stringent conditions, and the amino acid sequence has or partially has the function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 16, and the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions. A biomaterial, wherein the biomaterial comprises at least one of the following _b1 ) to _b3 ): _b1 ) a polynucleotide encoding the chimeric antigen receptor according to claim 9; _b2 ) a vector containing the polynucleotide described in _b1 ); _b3 ) A cell containing the vector described in _b2 ). A composition comprising the recombinant immune cell according to any one of claims 1 to 8, the chimeric antigen receptor according to claim 9 and/or the biomaterial according to claim 10; and, optionally, a pharmaceutically acceptable carrier. Use of the recombinant immune cell according to any one of claims 1 to 8, the chimeric antigen receptor according to claim 9 and/or the biomaterial according to claim 10 in the preparation of a medicament for treating and/or preventing a disease or condition; Wherein, the disease or condition is selected from inflammatory diseases or allergic diseases mediated by type 2 immune response, and diseases with eosinophils as effector cells; Optionally, the inflammatory disease or allergic disease mediated by the type 2 immune response includes one or more of asthma, allergic rhinitis, inflammatory skin disease, and food allergy; Optionally, the diseases with eosinophils as effector cells include one or more of acute and chronic asthma, eosinophilia, nasal polyps caused by eosinophils, enteritis caused by eosinophils, eosinophilic dermatitis, chronic obstructive pulmonary disease, and eosinophilic leukemia. Use of the recombinant immune cell according to any one of claims 1 to 8 as a carrier for delivering biological molecules for treating diseases. The method for preparing a recombinant immune cell according to any one of claims 1 to 8, comprising: (i) a step of introducing a construct for adoptive cell therapy into immune cells; and, (ii) a step of introducing a gene regulatory system into immune cells; and, optionally, (iii) A step of introducing biomolecules for treating diseases into immune cells.