Construction and use of CD19-targeting car-gdt cell

Abstract

A CD19-targeting CAR-gdT cell and a use thereof in B-cell tumors and autoimmune diseases. Specifically, the human CD19-targeting CAR-gdT cell comprises CD19-targeting scFv, a CD28 costimulatory signaling molecule, a CD3ζ domain, and exogenous IL-2 required for sustained gdT proliferation. In-vitro and in-vivo experiments show that the CAR-gdT cell exhibits significant specific killing of CD19-positive (CD19+) tumor cell strains, and no gdT cells with tumor-promoting properties emerge. The CD19-targeting CAR-gdT cell can be used as an allogeneic off-the-shelf therapy for targeting CD19+ tumor cells, and can also be used for treating autoimmune diseases caused by B cell hyperactivation.

Description

Construction and application of CAR-gdT cells targeting CD19 Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular 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 in which the patient's own immune system attacks its own healthy cells and tissues. Systemic lupus erythematosus (SLE) is an autoimmune inflammatory connective tissue disease that affects multiple organs and is common in young women. Early, mild, and atypical cases are increasing. The cause of this disease has not yet been confirmed, but a large number of studies have shown that genetics, environment, abnormal immune regulation, hormones, and some epigenetics are related to the onset of the disease. Among them, excessive B cell activation is a common feature of SLE patients, and CD19 is a biomarker for B cells that is expressed at all stages of B cell development. Therefore, cellular 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 treatment of hematologic malignancies. However, autologous CAR-T cell therapy faces significant challenges. Its limitations include: 1. Customized products are costly, significantly reducing patient accessibility; 2. Long preparation cycles, requiring 2-3 weeks, are associated with the fact that most patients receiving CAR-T therapy are at the end of their treatment cycle and experience rapid disease progression, making preparation too late; and 3. Some patients, due to various factors, such as poor health and multiple lines of prior treatment, are unable to provide T cells of sufficient quality or quantity to meet preparation requirements.

[0004] In order to improve patient accessibility and allow more patients to benefit, the development of allogeneic universal CAR-T products is a key strategy to address this problem. γδT cells in the T cell subset are non-MHC restricted and are considered to be useful for the preparation of allogeneic universal engineered T cells. However, the proportion of γδT cells in the periphery is extremely low, accounting for only 1%-5%. The enrichment and expansion of γδT cells and the transfection rate of CAR limit the preparation and application of engineered γδT cells. In addition, literature reports that there is a subtype of γδT cells that releases IL-17A, which is believed to promote tumorigenesis. Therefore, the safety of engineered γδT cells remains to be verified.

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

[0006] The purpose of the present invention is to provide a method for developing allogeneic universal, efficient and safe CAR-γδT cells targeting CD19.

[0007] In a first aspect of the present invention, an engineered T cell is provided, wherein the engineered T cell 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 the following formula I: CAR-LX (I)

[0009] in,

[0010] CAR is the coding sequence of the chimeric antigen receptor;

[0011] L is none or a linker coding sequence;

[0012] X is the interleukin 2 (IL-2) coding sequence.

[0013] In another preferred embodiment, the linker is a cleavable connecting peptide.

[0014] In another preferred embodiment, the cleavable connecting peptide is a self-cleaving 2A peptide, preferably a T2A peptide.

[0015] In another preferred embodiment, the IL-2 is mature IL-2, full-length IL-2, or an active fragment thereof.

[0016] In another preferred embodiment, the amino acid sequence of IL-2 is shown in SEQ ID NO: 7.

[0017] In another preferred embodiment, the sequence of the polynucleotide is shown as SEQ ID NO: 8.

[0018] In another preferred embodiment, the structure of the chimeric antigen receptor (CAR) is shown in the following formula II: L-scFv-H-TM-C-CD3ζ (II)

[0019] in,

[0020] "-" each independently represents nothing or a linker peptide.

[0021] L is none or a signal peptide sequence;

[0022] scFv is a CD19-targeting scFv;

[0023] H is the hinge region;

[0024] TM is the transmembrane domain;

[0025] C is no or co-stimulatory signaling molecules;

[0026] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0027] 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.

[0028] In another preferred embodiment, the L is a GM-CSF receptor signal peptide sequence.

[0029] In another preferred embodiment, the sequence of L is shown in SEQ ID NO: 1.

[0030] In another preferred embodiment, the scFv comprises a heavy chain variable region and a light chain variable region.

[0031] In another preferred embodiment, the heavy chain variable region and light chain variable region of the scFv are derived from a humanized antibody.

[0032] In another preferred embodiment, the sequence of the scFv is shown in SEQ ID NO: 2.

[0033] In another preferred embodiment, the H is a hinge region selected from the group consisting of Fc, CD8, CD28, CD137, or a combination thereof.

[0034] In another preferred embodiment, the H is a hinge region derived from IgG4 Fc.

[0035] In another preferred embodiment, the sequence of H is shown in SEQ ID NO: 3.

[0036] 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.

[0037] In another preferred embodiment, the TM is a transmembrane region derived from CD28.

[0038] In another preferred embodiment, the C is a co-stimulatory signal molecule of a protein that is absent or selected from the following group: 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.

[0039] In a second aspect of the present invention, a method for preparing the engineered T cells according to the first aspect of the present invention is provided, the method comprising the following steps:

[0040] (a) providing γδT cells to be modified; and

[0041] (b) transducing the polynucleotide or vector containing the chimeric antigen receptor and the exogenous IL-2 coding sequence into the γδ T cells, thereby obtaining the engineered T cells.

[0042] In another preferred embodiment, the method further comprises the step of performing functional and effectiveness testing on the obtained engineered immune cells.

[0043] 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.

[0044] In another preferred embodiment, the vector is selected from the following group: plasmid, viral vector.

[0045] In another preferred embodiment, the vector is in the form of a viral particle.

[0046] In another preferred embodiment, the vector is a lentiviral vector.

[0047] In the third aspect of the present invention, a preparation is provided, which contains the engineered T cells described in the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0048] In another preferred embodiment, the preparation is a liquid preparation.

[0049] In another preferred embodiment, the preparation is in the form of an injection.

[0050] 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.

[0051] In another preferred embodiment, the preparation further comprises a second anti-tumor active ingredient, preferably a second antibody or a chemotherapeutic agent.

[0052] In a fourth aspect of the present invention, there is provided a use of the engineered T cells described in the first aspect of the present invention for preparing a drug or preparation for preventing and / or treating a disease, wherein the disease is cancer or tumor, autoimmune disease, or a combination thereof.

[0053] In another preferred embodiment, the tumor is a CD19-positive tumor, preferably a tumor with high expression of CD19.

[0054] In another preferred embodiment, the tumor is a B cell tumor.

[0055] In another preferred embodiment, the tumor is selected from the group consisting of acute B-lymphocytic leukemia (B-ALL), non-Hodgkin's lymphoma, Hodgkin's lymphoma, and diffuse large B-cell lymphoma.

[0056] In another preferred embodiment, the autoimmune disease is a B cell-related autoimmune disease.

[0057] In another preferred embodiment, the autoimmune disease is selected from the following group: 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 Guillain syndrome (pSS), autoimmune hemolytic anemia, immunoglobulin G4-related disease, primary antiphospholipid antibody syndrome (pAPS), anti-neutrophil cytoplasmic antibody (ANCA)-associated vascular peptide (AAV), autoimmune thyroid disease (Graves' disease, GD), and autoimmune hepatitis (AIH).

[0058] In the fifth aspect of the present invention, a method for treating a disease is provided, comprising administering an appropriate amount of the engineered T cells of the first aspect of the present invention or the preparation of the third aspect of the present invention to a subject in need of treatment.

[0059] In another preferred embodiment, the subject is a human or a mammal.

[0060] In another preferred embodiment, the disease is selected from the group consisting of cancer or tumor, autoimmune disease, or a combination thereof.

[0061] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIG1 shows a schematic structural diagram of TAA09-AB-CAR and TAA09-CD-CAR of the present invention.

[0063] Figure 2 shows the positive infection rate of CAR-T cells in each group.

[0064] FIG3 shows the expression of CD19 on the cell surface of different tumor cell lines detected by flow cytometry.

[0065] FIG4 shows the cytotoxicity of UTAA09-AB-CAR-gdT and TAA09-AB-CAR-T cells.

[0066] FIG5 shows the cytotoxicity and cell proliferation results after each antigen stimulation in the repeated antigen stimulation experiment.

[0067] FIG6 shows the results of analyzing the tumor-promoting properties of gdT cells under repeated antigen stimulation conditions.

[0068] Figure 7 shows the in vivo efficacy evaluation results of UTAA09-AB-CAR-gdT and UTAA09-CD-CAR-gdT in B cell tumor animal models. DETAILED DESCRIPTION

[0069] After extensive and in-depth research, the inventors have provided a CD19-targeting allogeneic universal CAR-gdT cell. The CAR-gdT of the present invention has significant specific cytotoxicity against CD19-high-expressing tumor cells and has a persistent killing ability; at the same time, the CAR-gdT of the present invention co-expresses exogenous IL2 and has a sustained amplification ability; in vivo experiments in mice have demonstrated that the CAR-gdT of the present invention has significant and persistent anti-tumor activity and good safety. Experiments have shown that the CAR-gdT cells of the present invention unexpectedly have stronger and more persistent tumor cell killing ability and higher safety compared to αβT cells expressing the same CAR. The present invention was completed on this basis.

[0070] γδ T cells

[0071] T cells are divided into different types of subsets. Unlike αβT cells, γδT cells (also known as gdT cells) are non-MHC restricted.

[0072] The anti-tumor mechanisms of γδT cells include: (1) γδT cells induce apoptosis or death of tumor cells through two pathways (perforin lysozyme and death receptor pathways) or by inducing related apoptotic ligands; (2) secretion of interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) to indirectly exert anti-tumor effects; (3) γδT cells can mediate helper T cell (Treg) tumor immune responses by enhancing the antigen presentation function of dendritic cells; (4) γδT cells can inhibit the growth of tumor cells by exerting their cytotoxic effects. Compared with αβT cells, γδT cells are more suitable for the development of allogeneic universal engineered cells.

[0073] Human γδT cells can be divided into three distinct subsets: Vδ1, Vδ2, and Vδ3. The δ1 subset is abundant in mucosal epithelial tissues, accounting for approximately 10% to 15% of γδT cells and playing a key role in mucosal immunity. The δ2 ​​subset is primarily concentrated in peripheral blood, accounting for approximately 50% to 90% of γδT cells and representing the predominant circulating γδT lymphocyte population in healthy adults.

[0074] Type 1 Vδ1T cells (Vδ1 for short) are rare 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 active homing capabilities, and their infiltration in solid tumor tissues has been shown to be closely related to patients' good prognosis. 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 powerful innate immunity similar to that of NK cells, and therefore has excellent anti-tumor immunotherapy escape ability caused by heterogeneity. Unlike traditional αβT cells, Vδ1TCR is not restricted by the histocompatibility complex (MHC), thereby avoiding the occurrence of graft-versus-host disease (GVHD).

[0075] In the preparation of the allogeneic universal CAR-T cells of the present invention, γδT cells were used to construct CAR-γδT cells targeting CD19 (CAR-gdT cells of the present invention).

[0076] CAR of the present invention

[0077] 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: L-scFv-H-TM-C-CD3ζ (II)

[0078] in,

[0079] "-" each independently represents nothing or a linker peptide.

[0080] L is nothing or a signal peptide sequence; preferably, L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0081] The scFv is a CD19-targeting scFv; the preferred scFv sequence is shown in SEQ ID NO: 2.

[0082] H is a hinge region, preferably a hinge region derived from IgG4 Fc.

[0083] TM is a transmembrane domain; preferably, it is a transmembrane region derived from CD28.

[0084] C is no or co-stimulatory signaling molecules;

[0085] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0086] In a preferred embodiment, a CAR comprising the following elements is constructed: a signal peptide sequence derived from the macrophage colony-stimulating factor receptor (GM-CSF receptor) signal peptide sequence, a CD19-targeting scFv sequence derived from the humanized antibody sequence of the CD19 antibody clone FMC63, a hinge region sequence derived from the human IgG4 Fc sequence, a transmembrane region sequence derived from the transmembrane region sequence of the human CD28 molecule, and an intracellular region sequence comprising 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). The humanized CD19 scFv sequence is used to target and recognize the CD19 antigen on tumor cells; the human Fc hinge region serves to facilitate the formation of a complete spatial conformation of the humanized CD19 scFv antibody on the cell membrane, ensuring normal recognition of the CD19 antigen on the surface of tumor cells; the CD28 transmembrane region serves to enable the CD19-CAR protein to be properly anchored to the cell membrane; the human CD3ζ sequence serves as the first signal for T cell activation; and the human CD28 intracellular sequence serves as the second signal for T cell activation (a co-stimulatory molecule).

[0087] In a preferred example, the coding sequence of the CAR is shown as SEQ ID NO: 8.

[0088] Polynucleotides of the present invention

[0089] The present invention also provides a polynucleotide encoding the CAR of the present invention, wherein the polynucleotide further encodes interleukin 2 (IL-2).

[0090] In a preferred embodiment, the structure of the polynucleotide is shown in Formula I below: CAR-LX (I)

[0091] in,

[0092] CAR is the coding sequence of the chimeric antigen receptor;

[0093] L is none or a linker coding sequence;

[0094] X is interleukin 2 (IL-2).

[0095] In a preferred embodiment, the linker is a cleavable peptide linker. Preferably, the cleavable peptide linker is a self-cleaving 2A peptide, and more preferably, a T2A peptide.

[0096] In a preferred embodiment, the interleukin-2 is mature IL-2, full-length IL-2, or an active fragment thereof.

[0097] In a preferred example, the coding sequence of the interleukin is shown as SEQ ID NO: 7.

[0098] CAR-gdT cells of the present invention

[0099] As used herein, the terms "CAR-gdT of the present invention," "allogeneic universal CAR-T of the present invention," and "CAR-γδT of the present invention" are used interchangeably and refer to γδT cells expressing the CAR of the present invention, or γδT cells having the polynucleotide of the present invention integrated into their genome.

[0100] The CAR-gdT cells of the present invention can express the CD19-targeting CAR of the present invention, thereby having 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 CAR, wherein IL-2 is beneficial to improving the expansion of CAR-gdT cells and the durability of anti-tumor effects.

[0101] application

[0102] The present invention also provides applications of the allogeneic universal CAR-gdT cells of the present invention, which relate to multiple fields such as the diagnosis and treatment of CD19-related diseases, basic medical research, and biological research. A preferred application is for the treatment of CD19-related tumors and / or autoimmune diseases.

[0103] Generally, cells activated and expanded as described herein can be used to treat and prevent diseases such as tumors, autoimmune diseases, etc. Therefore, the present invention provides a method for treating a disease, which comprises administering a therapeutically effective amount of the CAR-gdT cells of the present invention to a subject in need thereof.

[0104] In one embodiment, the CAR-gdT cells of the present invention can undergo stable in vivo expansion and can persist for months to years. In addition, 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 tumor cells that express a high level of the antigen recognized by the CAR antigen binding domain. For example, the CAR-T cells of the present invention induce a specific immune response against B cells that express a high level of CD19.

[0105] Specifically, the types of diseases treated with the CAR of the present invention include, but are not limited to, tumors or cancers, autoimmune diseases, or a combination thereof.

[0106] 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 compositions of the present invention may include a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0107] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount 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 may be determined by clinical trials.

[0108] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, degree of infection or metastasis, and condition. The pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 7 The T cell composition can be administered at a dose of 10 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 using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a specific patient can be easily determined by a person skilled in the art of medicine by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0109] Administration of the subject composition can be carried out in any convenient manner, including by nebulization, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally.

[0110] 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 with (e.g., before, simultaneously, or after) any number of related treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or efavirenz treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0111] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration may be implemented according to practices accepted in the art. Typically, 1×10 5 to 1×10 10 The modified T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0112] The main advantages of the present invention include:

[0113] (a) The CAR-gdT cells of the present invention are non-MHC-restricted and allogeneic universal CAR-T cells.

[0114] (b) The CAR-gdT cells co-expressing exogenous IL-2 of the present invention have a long-lasting expansion capacity.

[0115] (c) The CAR-gdT cells of the present invention have significant in vitro and in vivo killing effects on cell lines expressing CD19.

[0116] (d) The results of in vivo experiments in mice showed that the CAR-gdT cells constructed by the present invention have high safety.

[0117] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed 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 as recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are by weight.

[0118] The sequence information involved in the examples is shown in Table 1.

[0119] Table 1 Sequence

[0120] Example 1: CAR molecule design and CAR-T cell preparation

[0121] In this embodiment, two new CAR structures targeting CD19 were first designed, as shown in Figure 1, respectively named TAA09-AB-CAR (as shown in Figure 1A) and TAA09-CD-CAR (as shown in Figure 1B). 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 underline is a connecting short peptide, the heavy chain variable region before the underline, and the light chain variable region after the underline), a hinge region sequence (SEQ ID NO: 3), a transmembrane region sequence (SEQ ID NO: 4), and a signal molecule for T cell activation (SEQ ID NO: 5-6). TAA09-AB-CAR also includes an IL2 sequence connected to a T2A peptide (SEQ ID NO: 7), while TAA09-CD-CAR does not include an IL2 sequence. Except for the T2A peptide and IL2 sequence, the other sequences of TAA09-AB-CAR and TAA09-CD-CAR are the same.

[0122] The designed CD19-targeting CAR gene was commissioned for gene synthesis at Suzhou Genewise Biotechnology Co., Ltd. A Kozak sequence was added to the front of the CD19-targeting CAR sequence, with restriction sites (XbaI / NotI) added at both ends. The sequence was then subcloned into a lentiviral expression plasmid (pCDH-CMV-MCS-KANA) with XbaI / NotI restriction sites. The constructed plasmids were named TAA09-AB-CAR plasmid and TAA09-CD-CAR plasmid, respectively.

[0123] CAR-T cells and CAR-gdT cells expressing the two aforementioned CAR constructs were generated, resulting in the following four engineered cell lines: UTAA09-AB-CAR-gdT, UTAA09-CD-CAR-gdT, TAA09-AB-CAR-T, and TAA09-CD-CAR-T. gdT represents γδT cells, and T represents αβT cells. The transfection efficiencies of the four engineered cell lines are shown in Figure 2.

[0124] Example 2: Cytotoxicity experiment of CAR-T cells

[0125] The expression of CD19 on the cell surface of four different tumor cell lines was analyzed. The four tumor cell lines were Wt Raji (wild-type Raji that naturally expresses human CD19 protein), Raji-CD19KO (Raji cells with CD19 knocked out), Wt K562 (wild-type K562 cells) and K562-CD19 cells (K562 cells that overexpress human CD19). The expression of CD19 in the four cells was detected by flow cytometry. The results are shown in Figure 3. Wt Raji cells and K562-CD19 cells are CD19 positive, while Raji-CD19KO cells and Wt K562 cells basically do not express CD19.

[0126] The cytotoxicity of UTAA09-AB-CAR-gdT and TAA09-AB-CAR-T on tumor cells was detected.

[0127] The results are shown in Figure 4. Compared with control gdT cells, UTAA09-AB-CAR-gdT exhibited significant specific cytotoxicity against CD19-positive cell lines Raji and K562-CD19, but had no specific killing effect on CD19-negative Raji-CD19KO cells and K562 cells. In addition, compared with TAA09-CAR-T cells, UTAA09-AB-CAR-gdT exhibited stronger cytotoxicity than TAA09-AB-CAR-T in a single killing process.

[0128] Example 3: In vitro persistence experiment of CAR-T cells killing

[0129] Using repeated antigen stimulation experiments, as shown in Figure 5A , the cytotoxicity, effector cell expansion, and tumor-promoting characteristics after each antigen stimulation were compared.

[0130] The cytotoxicity results are shown in Figure 5B. UTAA09-AB-CAR-gdT cells maintained robust anti-tumor activity after repeated antigen stimulation, similar to the anti-tumor effect of TAA09-AB-CAR-T cells. Under in vitro killing conditions, compared with UTAA09-CD-CAR-gdT cells without IL-2, the sustained cytotoxicity of UTAA09-AB-CAR-gdT cells carrying IL2 showed no significant difference.

[0131] The expansion results are shown in Figure 5C. The statistical results show that the IL2-carrying UTAA09-AB-CAR-gdT showed a similar expansion trend to the non-IL2-carrying TAA09-CD-CAR-T, with no statistical difference between the two. While the IL2-carrying TAA09-AB-CAR-T group had the strongest expansion capacity, excessive CAR-T cell expansion can lead to safety risks such as CRS in vivo, making UTAA09-AB-CAR-gdT more secure.

[0132] In addition, the UTAA09-CD-CAR-gdT group that does not carry IL2 has the weakest expansion ability. After the introduction of exogenous IL-2 structure, on the one hand, the expansion ability of CAR-gdT is improved, and on the other hand, the said increase is within an appropriate range, thus avoiding the safety risks such as CRS caused by excessive expansion ability in the body.

[0133] Example 4: Analysis of tumor-promoting properties of gdT cells under repeated antigen stimulation

[0134] According to literature reports, there is a subtype of gdT cells that releases IL-17A, which is believed to promote tumorigenesis. Based on this, the present invention continuously monitored the release of IL-17A under repeated antigen stimulation in vitro.

[0135] The results are shown in Figure 6 and Table 2. As can be seen from the results, no obvious IL-17A release was detected in TAA09-AB-CAR-gdT and TAA09-CD-CAR-gdT cells after 5 rounds of repeated antigen stimulation, which was lower than the type using αβT cells, indicating that the CAR-gdT cells of the present invention have high safety.

[0136] Table 2 Detection of IL-17A release under multiple rounds of stimulation (pg / mL)

[0137] Example 5: In vivo efficacy and safety of CAR-gdT cells

[0138] Based on the in vitro confirmation of the efficacy and safety of UTAA09-AB-CAR-gdT, we further verified its in vivo anti-tumor effect and safety in the B-NDG tumor-bearing mouse model (Raji tumor model).

[0139] The experimental process is shown in Figure 7A. Two days after intravenous inoculation of Raji-Luc-GFP cells, 18 female NPG mice were randomly divided into three groups based on body weight: a PBS group (5 mice), a gdT cell control group (5 mice), a UTAA09-AB-CAR-gdT group (5 mice), and a UTAA09-CD-CAR-gdT group (3 mice). All animals received a single injection via the tail vein, and changes in tumor burden were monitored in real time.

[0140] The experimental results are shown in Figures 7B-7E. The data show that mice infused with UTAA09-AB-CAR-gdT experienced a significant reduction in tumor burden, while all mice in the PBS and gdT control groups died by Day 19 due to excessive tumor burden (Figures 7B and 7C). This demonstrates that UTAA09-AB-CAR-gdT has significant anti-tumor activity and the ability to sustain tumor killing.

[0141] It is worth noting that the survival of 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 a relatively important role in maintaining the anti-tumor effect and persistence of CAR-gdT. 40% of the mice in the experimental group (UTAA09-AB-CAR-gdT group) were still in a tumor-free state at the end of the experiment, achieving complete elimination of the tumor. Statistics of mouse survival found that the overall survival of mice in the UTAA09-AB-CAR-gdT group was significantly prolonged (p < 0.01, Figure 7D), and the mice did not experience significant weight loss throughout the experiment (Figure 7E), indicating its good safety.

[0142] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

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 2, wherein The sequence of the polynucleotide is as shown in SEQ ID NO:

8.

5. The engineered T cell according to claim 1, wherein The structure of the chimeric antigen receptor 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.

6. The engineered T cell according to claim 5, wherein The sequence of the scFv is as shown in SEQ ID NO:

2.

7. A method for preparing the engineered T cells according to claim 1, characterized in that, The method comprises the following steps: (a) providing γδ T cells to be engineered; 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.

8. The method according to claim 7, wherein 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.

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

10. Use of the engineered T cells as claimed in claim 1 for the preparation of a drug or a 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.

11. The use according to claim 10, wherein, The tumor is a B cell tumor.

12. The use according to claim 11, wherein The tumor is selected from the group consisting of: acute B lymphoblastic leukemia (B-ALL), non-Hodgkin lymphoma, Hodgkin lymphoma, diffuse large B cell lymphoma.

13. The use according to claim 10, characterized in that, In another preferred embodiment, the autoimmune disease is a B cell-related autoimmune disease.

14. The use according to claim 13, wherein, 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), autoimmune hepatitis (AIH).

15. 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 9 to a subject in need of treatment.

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