Construction and application of CAR-gdT cell targeting CD19

Abstract

The invention provides a CAR-gdT cell targeting CD19 and an application of the CAR-gdT cell in B cell tumors and autoimmune diseases. Specifically, the CAR-gdT cell targeting the human CD19 disclosed by the invention comprises scFv targeting the CD19, a CD28 costimulatory signal molecule, a CD3 zeta structural domain and an exogenous IL-2 (Interleukin-2) required by continuous proliferation of the gdT. In-vitro and in-vivo experiments show that the CAR-gdT cell disclosed by the invention has obvious specific killing effect on CD19 positive CD19 + tumor cell strains, and no gdT cell with tumor promoting characteristic appears, so that the CAR-gdT cell has high safety. The CD19-CAR-gdT cell disclosed by the invention can be used as a novel allogenic universal therapy for targeting CD19 < + > tumor cells, and also provides a new strategy for treating autoimmune diseases caused by excessive activation of B cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the construction and application of CAR-gdT cells targeting CD19. Background Art

[0002] Systemic lupus erythematosus, also known as SLE or lupus, is a chronic disease mainly characterized by the patient's own immune system attacking their own healthy cells and tissues. Systemic lupus erythematosus (SLE) is an autoimmune inflammatory connective tissue disease that affects multiple organs and is more common in young women. The number of early, mild, and atypical cases is increasing day by day. The cause of this disease has not been confirmed yet. A large number of studies have shown that genetics, environment, abnormal immune regulation, hormones, and some epigenetic factors are related to the onset of this disease. Among them, overactivation of B cells is a common feature of SLE patients, and CD19 is a biomarker of B cells and is expressed at all stages of B cell development. Therefore, cell immunotherapy targeting CD19 has the potential to be applied to the treatment of B cell-related autoimmune diseases such as systemic lupus erythematosus.

[0003] Chimeric antigen receptor T cell therapy (CAR-T) has achieved remarkable clinical efficacy in the field of blood tumor treatment. However, autologous CAR-T cell drugs face huge challenges, and their limitations mainly include: 1. Customized products with high costs, which greatly reduce the accessibility of patients; 2. Long preparation cycles, which take 2-3 weeks, and most patients receiving CAR-T therapy are end-stage patients with rapid disease progression and may not have enough time for preparation; 3. Some patients are unable to provide T cells with sufficient quality or quantity to meet the preparation requirements due to reasons such as their physical health level and excessive previous lines of treatment.

[0004] To improve patient accessibility and benefit more patients, developing allogeneic universal CAR-T products is a key strategy to solve this problem. Gamma-delta T cells in the T cell subset are non-MHC restricted and are considered suitable for the preparation of allogeneic universal engineered T cells. However, the proportion of gamma-delta T cells in the periphery is extremely low, only accounting for 1%-5%. The enrichment, amplification, and transfection rate of CAR of gamma-delta T cells limit the preparation and application of engineered gamma-delta T cells. In addition, the literature reports that there is a subtype of gamma-delta T cells that releases IL-17A, and this subtype is considered likely to promote tumorigenesis. Therefore, the safety of engineered gamma-delta T cells needs to be verified.

[0005] Therefore, there is an urgent need in this field to develop allogeneic universal, highly efficient, and safe CAR-gamma-delta T cells targeting CD19. Summary of the Invention

[0006] The object of the present invention is to develop allogeneic universal, highly efficient, and safe CAR-gamma-delta T cells targeting CD19.

[0007] In the first aspect of the present invention, there is provided an engineered T cell that co-expresses a chimeric antigen receptor targeting CD19 and exogenous interleukin-2 (IL-2), and the T cell is a γδ T cell.

[0008] In another preferred embodiment, the chimeric antigen receptor and the exogenous interleukin-2 are encoded by a polynucleotide, and the structure of the polynucleotide is shown in Formula I below:

[0009] CAR-L-X (I)

[0010] Wherein,

[0011] CAR is the coding sequence of the chimeric antigen receptor;

[0012] L is none or a linker coding sequence;

[0013] X is the interleukin-2 (IL-2) coding sequence.

[0014] In another preferred embodiment, the linker is a cleavable linker peptide.

[0015] In another preferred embodiment, the cleavable linker peptide is a self-cleaving 2A peptide; preferably, it is the T2A peptide.

[0016] In another preferred embodiment, the IL-2 is mature IL-2, full-length IL-2, or an active fragment thereof.

[0017] In another preferred embodiment, the amino acid sequence of the IL-2 is as shown in SEQ ID NO: 7.

[0018] In another preferred embodiment, the sequence of the polynucleotide is as shown in SEQ ID NO: 8.

[0019] In another preferred embodiment, the structure of the chimeric antigen receptor (CAR) is shown in Formula II below:

[0020] L-scFv-H-TM-C-CD3ζ (II)

[0021] Wherein,

[0022] Each "-" is independently none or a linker peptide.

[0023] L is none or a signal peptide sequence;

[0024] scFv is an scFv targeting CDl9;

[0025] H is a hinge region;

[0026] TM is a transmembrane domain;

[0027] C is a co-stimulatory signal molecule or none;

[0028] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0029] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of: GM-CSF receptor, CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0030] In another preferred embodiment, the L is the GM-CSF receptor signal peptide sequence.

[0031] In another preferred embodiment, the sequence of the L is as shown in SEQ ID NO: 1.

[0032] In another preferred embodiment, the scFv comprises a heavy chain variable region and a light chain variable region.

[0033] In another preferred embodiment, the heavy chain variable region and the light chain variable region of the scFv are derived from a humanized antibody.

[0034] In another preferred embodiment, the sequence of the scFv is as shown in SEQ ID NO: 2.

[0035] In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of: Fc, CD8, CD28, CD137, or a combination thereof.

[0036] In another preferred embodiment, the H is a hinge region derived from IgG4 Fc.

[0037] In another preferred embodiment, the sequence of the H is as shown in SEQ ID NO: 3.

[0038] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0039] In another preferred embodiment, the TM is a transmembrane region derived from CD28.

[0040] In another preferred embodiment, C is a co-stimulatory signaling molecule that is absent or selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0041] In a second aspect of the present invention, there is provided a method for preparing the engineered T cells described in the first aspect of the present invention, the method comprising the following steps:

[0042] (a) providing γδ T cells to be modified; and

[0043] (b) transducing the polynucleotide or vector containing the chimeric antigen receptor and the exogenous IL-2 coding sequence into the γδ T cells to obtain the engineered T cells.

[0044] In another preferred embodiment, the method further comprises the step of detecting the function and effectiveness of the obtained engineered immune cells.

[0045] In another preferred embodiment, the vector is selected from the group consisting of: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, adeno-associated viral vector (AAV), retroviral vector, transposon, or a combination thereof.

[0046] In another preferred embodiment, the vector is selected from the group consisting of: plasmid, viral vector.

[0047] In another preferred embodiment, the vector is in the form of viral particles.

[0048] In another preferred embodiment, the vector is a lentiviral vector.

[0049] In a third aspect of the present invention, there is provided a preparation containing the engineered T cells described in the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0050] In another preferred embodiment, the preparation is a liquid preparation.

[0051] In another preferred embodiment, the dosage form of the preparation is an injection.

[0052] In another preferred embodiment, the concentration of the engineered T cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0053] In another preferred embodiment, the preparation further comprises a second anti-tumor active ingredient, preferably including a second antibody or a chemotherapeutic agent.

[0054] In a fourth aspect of the present invention, there is provided a use of the engineered T cell as described in the first aspect of the present invention for preparing a drug or a preparation for preventing and / or treating a disease, wherein the disease is cancer or tumor, an autoimmune disease, or a combination thereof.

[0055] In another preferred embodiment, the tumor is a CD19-positive tumor, preferably a tumor with high CD19 expression.

[0056] In another preferred embodiment, the tumor is a B cell tumor.

[0057] In another preferred embodiment, the tumor is selected from the group consisting of acute B lymphoblastic leukemia (B-ALL), non-Hodgkin lymphoma, Hodgkin lymphoma, and diffuse large B cell lymphoma.

[0058] In another preferred embodiment, the autoimmune disease is a B cell-related autoimmune disease.

[0059] In another preferred embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, type I diabetes, neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis (MG), rheumatoid arthritis (RA), multiple sclerosis, Hashimoto's thyroiditis (HT), pemphigus, systemic sclerosis, primary Sjögren's syndrome (pSS), autoimmune hemolytic anemia, immunoglobulin G4-related disease, primary antiphospholipid antibody syndrome (pAPS), antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), autoimmune thyroid disease (Graves' disease, GD), and autoimmune hepatitis (AIH).

[0060] In a fifth aspect of the present invention, there is provided a method for treating a disease, comprising administering an appropriate amount of the engineered T cell as described in the first aspect of the present invention or the preparation as described in the third aspect of the present invention to a subject in need of treatment.

[0061] In another preferred embodiment, the subject is a human or a mammal.

[0062] In another preferred embodiment, the disease is selected from the group consisting of cancer or tumor, an autoimmune disease, or a combination thereof.

[0063] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Shows the schematic diagrams of the structures of TAA09-AB-CAR and TAA09-CD-CAR of the present invention.

[0065] Figure 2 Shows the positive infection rates of each group of CAR-T cells.

[0066] Figure 3 Shows the expression of cell surface CD19 of different tumor cell lines detected by flow cytometry.

[0067] Figure 4 Shows the cytotoxicity of UTAA09-AB-CAR-gdT and TAA09-AB-CAR-T cells.

[0068] Figure 5 Shows the cytotoxicity and cell expansion results after each antigen stimulation in the antigen repeated stimulation experiment.

[0069] Figure 6 Shows the analysis results of the tumor-promoting characteristics of gdT cells under antigen repeated stimulation conditions.

[0070] Figure 7 Shows the in vivo efficacy evaluation results of UTAA09-AB-CAR-gdT and UTAA09-CD-CAR-gdT on a B-cell tumor animal model. Detailed implementation manners

[0071] Through extensive and in-depth research, the present inventors provide an allogeneic universal CAR-gdT cell targeting CD19. The CAR-gdT of the present invention has significant specific cytotoxicity against CD19-highly expressed tumor cells and has a persistent killing ability; meanwhile, the CAR-gdT of the present invention co-expresses exogenous IL2 and has a continuous expansion ability; in vivo experiments on mice prove that the CAR-gdT of the present invention has significant and persistent anti-tumor activity and has good safety. Experiments prove that, compared with αβT cells expressing the same CAR, the CAR-gdT cells of the present invention unexpectedly have stronger and more persistent tumor cell killing ability and higher safety. On this basis, the present invention is completed.

[0072] γδT cells

[0073] T cells are divided into different types of subsets. Different from αβT cells, γδT cells (also known as gdT cells) have non-MHC restriction.

[0074] The anti-tumor mechanisms of γδ T cells mainly include: (1) γδ T cells induce apoptosis or death of tumor cells through two pathways (perforin-lysosome and death receptor pathways) or by inducing related apoptosis ligands; (2) indirectly exert anti-tumor effects by secreting interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α); (3) γδ T cells can mediate the tumor immune response of helper T cells (Tregs) by enhancing the antigen presentation of dendritic cells; (4) γδ T cells can inhibit the growth of tumor cells by exerting their cytotoxic effects. Compared with αβ T cells, γδ T cells are more suitable for the development of allogeneic universal engineered cells.

[0075] Human γδ T cells can be divided into three different subsets: Vδ1, Vδ2, and Vδ3. The δ1 subset is rich in mucosal epithelial tissues, accounting for about 10% - 15% of γδ T cells, and plays an important role in mucosal immunity; the δ2 subset is mainly concentrated in peripheral blood, accounting for about 50% - 90% of γδ T cells, and is the main circulating γδ T lymphocytes in healthy adults.

[0076] Type 1 Vδ1 T cells (abbreviated as Vδ1) are scarce in the human peripheral system but are abundant immune cells in epithelial and mucosal tissues. Vδ1 cells naturally highly express chemokine receptors and chemokines, have the ability of active homing, and their infiltration in solid tumor tissues has been proven to be closely related to the good prognosis of patients. The TCR of Vδ1 cells is highly sensitive to antigen recognition and can broadly recognize phospholipid antigens induced by tumor stress signals. Moreover, it can directly kill tumor cells through its strong innate immunity similar to NK cells, so it has excellent anti-tumor immune therapy escape ability due to heterogeneity. Different from traditional αβ T cells, Vδ1 TCR is not restricted by the major histocompatibility complex (MHC), thus avoiding the occurrence of graft-versus-host disease (GVHD).

[0077] In the preparation of the allogeneic universal CAR-T cells of the present invention, γδ T cells are used to construct CAR-γδ T cells targeting CD19 (CAR-γδ T cells of the present invention).

[0078] The CAR of the present invention

[0079] In the present invention, a chimeric antigen receptor (CAR) is provided, which comprises an antigen-binding domain targeting CD19. The preferred structure of the CAR of the present invention is shown in Formula II:

[0080] L-scFv-H-TM-C-CD3ζ (II)

[0081] Wherein,

[0082] Each "-" is independently none or a linker peptide.

[0083] L is a signal peptide sequence that is absent or present; preferably, L is the signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0084] The scFv is an scFv targeting CD19; preferably, the scFv sequence is as shown in SEQ ID NO: 2.

[0085] H is a hinge region; preferably, it is a hinge region derived from IgG4 Fc.

[0086] TM is a transmembrane domain; preferably, it is a transmembrane region derived from CD28.

[0087] C is a costimulatory signal molecule that is absent or present;

[0088] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0089] In a preferred embodiment, a CAR comprising the following elements was constructed: the signal peptide sequence was from the macrophage colony-stimulating factor receptor (GM-CSF receptor) signal peptide sequence, the scFv sequence targeting CD19 was from the humanized antibody sequence of the CD19 antibody with clone number FMC63, the hinge region sequence was the human IgG4 Fc sequence, the transmembrane region sequence was the transmembrane region sequence of the human CD28 molecule, and the intracellular region sequence included the human CD3ζ sequence (the first signal for T cell activation) and the intracellular sequence of the human CD28 molecule (the second signal for T cell activation). Among them, the humanized CD19 scFv sequence was used to target and recognize the CD19 antigen on tumor cells; the function of the human Fc hinge region was to facilitate the humanized CD19 scFv antibody to form a complete spatial conformation on the cell membrane to ensure normal recognition of the CD19 antigen on the surface of tumor cells; the function of the CD28 transmembrane region was to enable the CD19-CAR protein to be normally anchored on the cell membrane; the human CD3ζ sequence was the first signal for T cell activation; the human CD28 intracellular sequence was the second signal for T cell activation (costimulatory molecule).

[0090] In a preferred embodiment, the coding sequence of the CAR is as shown in SEQ ID NO: 8.

[0091] The polynucleotide of the present invention

[0092] The present invention also provides a polynucleotide encoding the CAR of the present invention, and the polynucleotide also encodes interleukin 2 (IL-2).

[0093] In a preferred embodiment, the structure of the polynucleotide is as shown in Formula I below:

[0094] CAR-L-X (I)

[0095] Wherein,

[0096] CAR is the coding sequence of the chimeric antigen receptor;

[0097] L is none or the coding sequence of a linker;

[0098] X is interleukin-2 (IL-2).

[0099] In a preferred embodiment, the linker is a cleavable linker peptide. Preferably, the cleavable linker peptide is a self-cleaving 2A peptide; more preferably it is the T2A peptide.

[0100] In a preferred embodiment, the interleukin-2 is mature IL-2, full-length IL-2, or an active fragment thereof.

[0101] In a preferred embodiment, the coding sequence of the interleukin is as shown in SEQ ID NO: 7.

[0102] The CAR-gdT cells of the present invention

[0103] As used herein, the terms "the CAR-gdT of the present invention", "the allogeneic universal CAR-T of the present invention", "the CAR-yδT of the present invention" are used interchangeably, and all refer to γδT cells expressing the CAR of the present invention, or γδT cells in which the polynucleotide of the present invention is integrated into the genome.

[0104] The CAR-gdt cells of the present invention can express the CAR targeting CD19 of the present invention, and thus have a killing effect on cells with high CD19 expression; in addition, the CAR-gdT cells of the present invention co-express exogenous IL-2 while expressing the CAR, and IL-2 is beneficial to improving the expansion of CAR-gdT cells and the persistence of the anti-tumor effect.

[0105] Application

[0106] The present invention also provides the application of the allogeneic universal CAR-gdT cells of the present invention, which is involved in multiple fields such as the diagnosis and treatment of diseases related to CD19, basic medical research, biological research, etc. A preferred application is for the treatment of CD19-related tumors and / or autoimmune diseases.

[0107] Generally, the cells activated and amplified as described herein can be used for the treatment and prevention of diseases such as tumors and autoimmune diseases. Therefore, the present invention provides a method for treating a disease, which includes administering a therapeutically effective amount of the CAR-gdT cells of the present invention to a subject in need thereof.

[0108] In one embodiment, the CAR-gdT cells of the present invention can undergo stable in vivo expansion and persist for months to years. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, wherein the CAR-gdT cells can induce a specific immune response against highly expressed tumor cells of the antigen recognized by the CAR antigen-binding domain. For example, the CAR-T cells of the present invention elicit a specific immune response against CD19-highly expressed B cells.

[0109] Specifically, the types of diseases treatable with the CAR of the present invention include, but are not limited to: tumors or cancers, autoimmune diseases, or combinations thereof.

[0110] The CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention can include a population of target cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0111] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The quantity and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or can be determined by clinical trials.

[0112] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount", or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the patient (subject)'s age, weight, degree of infection or metastasis, and individual differences in the condition. A pharmaceutical composition comprising the T cells described herein can be administered at a dose of 10 4 to 10 9 cells / kg body weight, preferably at a dose of 10 5 to 10 7 cells / kg body weight (including all integer values within the range). The T cell composition can also be administered multiple times at these doses. The cells can be administered by infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by those skilled in the medical art by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0113] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection, or intraperitoneally to a patient.

[0114] In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, concurrently, or after) with any number of relevant treatment modalities, including but not limited to treatment with the following reagents: such reagents as antiviral therapies, cidofovir, cytarabine (also known as ARA-C), or treatment with natalizumab for MS patients or efalizumab for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the present invention can be used in combination with: chemotherapy, radiation, immunosuppressive agents, such as, cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination (e.g., before, concurrently, or after) with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In an additional embodiment, the expanded cells are administered before or after surgery.

[0115] The doses of the above treatments administered to a patient will vary with the precise nature of the disorder being treated and the recipient of the treatment. Dosage ratios for human administration can be practiced according to accepted practice in the art. Generally, for each treatment or course of treatment, 1×10 5 to 1×10 10 modified T cells of the present invention can be administered to a patient, for example, by intravenous infusion.

[0116] The main advantages of the present invention include:

[0117] (a) The CAR-gdT cells of the present invention are non-MHC restricted and are allogeneic universal CAR-T cells.

[0118] (b) The CAR-gdT cells of the present invention co-expressing exogenous IL-2 have persistent expansion ability.

[0119] (c) The CAR-gdT cells of the present invention have significant in vitro and in vivo killing effects on cell lines expressing CD19.

[0120] (d) The results of in vivo experiments in mice show that the CAR-gdT cells constructed in the present invention have high safety.

[0121] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0122] The sequence information involved in the embodiments is shown in Table 1.

[0123] Table 1 Sequences

[0124]

[0125] Example 1: CAR Molecule Design and CAR-T Cell Preparation

[0126] In this example, two novel CAR structures targeting CD19 were first designed, as Figure 1 shown, and named TAA09-AB-CAR (as Figure 1 A) and TAA09-CD-CAR (as Figure 1 B), respectively. Both CAR structures include a signal peptide sequence (SEQ ID NO: 1), a humanized CD19 scFv sequence targeting human CD19 (SEQ ID NO: 2, the underlined part is the linker peptide, the part before the underline is the heavy chain variable region, and the part after the underline is the light chain variable region), a hinge region sequence (SEQ ID NO: 3), a transmembrane region sequence (SEQ ID NO: 4), signal molecules for T cell activation (SEQ ID NOs: 5-6). TAA09-AB-CAR also contains an IL2 sequence (SEQ ID NO: 7) linked by a T2A peptide, while TAA09-CD-CAR does not contain the IL2 sequence. Except for the T2A peptide and the IL2 sequence, the other sequences of TAA09-AB-CAR and TAA09-CD-CAR are the same.

[0127] The designed CAR genes targeting CD19 were entrusted to Genewiz (Suzhou) Inc. for gene synthesis. A Kozak sequence was added in front of the CAR sequence targeting CD19, and restriction enzyme sites (XbaI / NotI) were added at both ends, and then subcloned into the lentiviral expression plasmid (pCDH-CMV-MCS-KANA). The subcloning restriction enzyme sites were XbaI / NotI. The constructed plasmids were named TAA09-AB-CAR plasmid and TAA09-CD-CAR plasmid, respectively.

[0128] Prepare CAR-T cells and CAR-gdT cells expressing the above two CAR structures, and obtain the following four engineered cells respectively: UTAA09-AB-CAR-gdT, UTAA09-CD-CAR-gdT, TAA09-AB-CAR-T, and TAA09-CD-CAR-T. Among them, gdT represents γδT cells, and T represents αβT cells. The transfection efficiencies of the four engineered cells are as Figure 2 shown.

[0129] Example 2: Cytotoxicity experiment of CAR-T cells

[0130] Analyze the expression of CD19 on the cell surface of four different tumor cell lines. The four tumor cell lines are WtRaji (wild-type Raji that naturally expresses human CD19 protein), Raji-CD19KO (Raji cells with CD19 knockout), Wt K562 (wild-type K562 cells), and K562-CD19 cells (K562 cells with overexpression of human CD19). Detect the CD19 expression of the four cells by flow cytometry. The results are as Figure 3 shown. Wt Raji cells and K562-CD19 cells are CD19 positive, while Raji-CD19KO cells and Wt K562 cells basically do not express CD19.

[0131] Detect the cytotoxicity of UTAA09-AB-CAR-gdT and TAA09-AB-CAR-T against tumor cells.

[0132] The results are as Figure 4 shown. Compared with control gdT cells, UTAA09-AB-CAR-gdT shows significant specific cytotoxicity against CD19-positive cell lines Raji and K562-CD19, but does not have specific killing against CD19-negative Raji-CD19KO cells and K562 cells; in addition, compared with TAA09-CAR-T cells, UTAA09-AB-CAR-gdT shows stronger cytotoxicity than TAA09-AB-CAR-T during a single killing process.

[0133] Example 3: Persistence experiment of in vitro killing of CAR-T cells

[0134] Use the antigen repeated stimulation experiment, as Figure 5 shown in A, to compare the cytotoxicity strength, the amplification of effector cells, and the analysis of pro-tumor characteristics after each antigen stimulation.

[0135] The cytotoxicity results are as Figure 5As shown in Figure B. After multiple antigen repeated stimulations, UTAA09-AB-CAR-gdT can still maintain strong anti-tumor activity, which is similar to the anti-tumor effect of TAA09-AB-CAR-T; under the in vitro killing system conditions, there is no significant difference in the continuous killing ability of target cells between UTAA09-AB-CAR-gdT cells carrying IL-2 and UTAA09-CD-CAR-gdT cells without IL-2.

[0136] The results of the amplification are as Figure 5 shown in Figure C. It can be seen from the statistical results that UTAA09-AB-CAR-gdT carrying IL-2 shows an amplification trend similar to that of TAA09-CD-CAR-T without IL-2, and there is no statistical difference between the two; while the amplification ability of the TAA09-AB-CAR-T group carrying IL-2 is the strongest, but too strong amplification ability of CAR-T cells will lead to safety risks such as CRS in vivo. Therefore, UTAA09-AB-CAR-gdT is safer.

[0137] In addition, the amplification ability of the UTAA09-CD-CAR-gdT group without IL-2 is relatively the weakest. After introducing the exogenous IL-2 structure, on the one hand, it improves the amplification ability of CAR-gdT, and on the other hand, the improvement amplitude is within a suitable range, thus avoiding the safety risks such as CRS caused by too strong amplification ability in vivo.

[0138] Example 4: Analysis of the tumor-promoting characteristics of gdT cells under antigen repeated stimulation conditions

[0139] According to literature reports, there is a subtype in gdT cells that releases IL-17A, and this subtype is considered to possibly promote tumorigenesis. Based on this, in the present invention, under the condition of in vitro antigen repeated stimulation, the release of IL-17A was continuously monitored.

[0140] The results are as Figure 6 shown in Figure and Table 2. It can be seen from the results that no obvious release of IL-17A was detected in TAA09-AB-CAR-gdT and TAA09-CD-CAR-gdT cells under 5 rounds of antigen repeated stimulation conditions, and both are lower than the type of αβT cells, indicating that the CAR-gdT cells of the present invention have high safety.

[0141] Table 2 Detection of IL-17A release amount under multiple rounds of stimulation (pg / mL)

[0142]

[0143] Example 5: In vivo efficacy and safety of CAR-gdT cells

[0144] On the basis of verifying the efficacy and safety of UTAA09-AB-CAR-gdT in vitro, we further verified its anti-tumor effect and safety in a B-NDG tumor-bearing mouse (Raji tumor model) model.

[0145] The experimental procedure is as Figure 7 shown in A. Two days after 18 female NPG mice were intravenously inoculated with Raji-Luc-GFP cells, they were randomly divided into 3 groups according to body weight screening: PBS group (5 mice), gdT cell control group (5 mice), UTAA09-AB-CAR-gdT group (5 mice), and UTAA09-CD-CAR-gdT group (3 mice). All animals were administered by single tail vein injection, and the changes in the tumor burden level of the mice were monitored in real time.

[0146] The experimental results are as Figure 7 shown in B-7E. It can be seen from the experimental data that in the mice in the UTAA09-AB-CAR-gdT group after reinfusion, the tumor burden of the mice was effectively alleviated. All the mice in the PBS and gdT control groups died due to excessive tumor burden on Day 19 ( Figure 7 B and Figure 7 C). This shows that UTAA09-AB-CAR-gdT has significant anti-tumor activity and the ability to continuously kill tumors.

[0147] It is worth noting that the survival period of the mice in the UTAA09-CD-CAR-gdT group was similar to that of the PBS and gdT groups, indicating that under in vivo environmental conditions, the presence of exogenous IL-2 plays an important role in maintaining the anti-tumor effect and persistence of CAR-gdT. 40% of the experimental group mice (UTAA09-AB-CAR-gdT group) were still in a tumor-free state at the end of the experiment, achieving complete tumor clearance. Statistical analysis of the mouse survival period found that in the mice in the UTAA09-AB-CAR-gdT group after reinfusion, the overall survival period was significantly prolonged (p < 0.01, Figure 7 D), and the mice did not show a significant decrease in body weight during the entire experiment ( Figure 7 E), indicating its good safety.

[0148] All the documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. An engineered T cell, characterized in that, The engineered T cells co-express a chimeric antigen receptor targeting CD19 and exogenous interleukin-2 (IL-2), and the T cells are γδ T cells.

2. The engineered T cell according to claim 1, wherein The chimeric antigen receptor and the exogenous interleukin-2 are encoded by a polynucleotide, and the structure of the polynucleotide is shown in the following formula I: CAR-L-X (I) Wherein, CAR is the coding sequence of the chimeric antigen receptor; L is none or a linker coding sequence; X is the interleukin-2 (IL-2) coding sequence.

3. The engineered T cell according to claim 1, wherein The exogenous interleukin-2 is mature IL-2, full-length IL-2, or an active fragment thereof.

4. The engineered T cell according to claim 1, wherein The structure of the chimeric antigen receptor (CAR) is shown in the following formula II: L-scFv-H-TM-C-CD3ζ (II) Wherein, "-" are each independently none or a linker peptide; L is none or a signal peptide sequence; scFv is an scFv targeting CD19; H is a hinge region; TM is a transmembrane domain; C is none or a co-stimulatory signaling molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

5. A method for preparing the engineered T cells according to claim 1, characterized in that, The method includes the following steps: (a) Providing γδ T cells to be modified; and (b) Transducing the polynucleotide or vector containing the coding sequences of the chimeric antigen receptor and the exogenous IL-2 into the γδ T cells to obtain the engineered T cells.

6. The method according to claim 5, characterized in that, The vector is selected from the group consisting of: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, adeno-associated viral vector (AAV), retroviral vector, transposon, or a combination thereof.

7. A preparation, characterized in that, The preparation contains the engineered T cells as claimed in claim 1, and a pharmaceutically acceptable carrier.

8. Use of the engineered T cells as claimed in claim 1 for preparing a drug or preparation for preventing and / or treating a disease, wherein the disease is selected from the group consisting of: cancer or tumor, autoimmune disease, or a combination thereof.

9. The use according to claim 8, wherein In another preferred example, the autoimmune disease is a B cell-related autoimmune disease.

10. A method for treating a disease, comprising administering an appropriate amount of the engineered T cells as claimed in claim 1 or the preparation as claimed in claim 7 to a subject in need of treatment.

Images