Preparation of Foxp3 metabolism regulation type CAR-T cell

Abstract

The invention provides preparation of Foxp3 metabolism regulation type CAR-T cells, and belongs to the technical field of cellular immunotherapy. The invention provides a fusion gene which comprises a coding gene of Foxp3 and a coding gene of a chimeric antigen receptor. Based on the fusion gene provided by the invention, a CAR-T cell of overexpressed Foxp3 can be constructed, so that a regulatory T cell metabolism mode (Treg cell) is simulated, and CAR-T cell metabolism is remodeled to improve a strategy of an anti-tumor effect. Through verification, compared with a traditional CAR-T cell, the Foxp3 metabolism regulation type CAR-T cell has a stronger and more lasting anti-tumor effect, and can play a long-term immune surveillance function.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell immunotherapy, and in particular relates to the preparation of Foxp3 metabolically regulated CAR-T cells. Background Art

[0002] Although CAR-T cell therapy has achieved unprecedented success in the treatment of hematological malignancies, the therapeutic effect of CAR-T cell therapy in solid tumors is still unsatisfactory. Among them, the low sugar, hypoxia, and high lactate environment of the tumor microenvironment severely limit the energy metabolism of CAR-T cells and weaken the effectiveness and durability of CAR-T cells. Reshaping CAR-T cell metabolism and overcoming the tumor microenvironment are effective means to improve the therapeutic effect of CAR-T cell therapy in solid tumors. Therefore, discovering a strategy based on metabolic reprogramming to improve the adaptation of CAR-T cells to the tumor microenvironment will help improve the immunotherapy effect of CAR-T cells in the treatment of solid tumors. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing metabolically regulated CAR-T cells, which can improve the metabolism of CAR-T cells and enhance the therapeutic effect of CAR-T cells in tumors by expressing the coding gene of Foxp3Foxp3.

[0004] The present invention provides a fusion gene, comprising a Foxp3 coding gene and a chimeric antigen receptor coding gene; the amino acid sequence of the Foxp3 is shown in SEQ ID NO.2.

[0005] As an optional solution, the nucleotide sequence of the Foxp3 encoding gene is shown in SEQ ID NO.1.

[0006] As an optional scheme, the chimeric antigen receptor includes but is not limited to a third-generation CAR or a second-generation CAR; the third-generation CAR includes a scFv sequence, a CD28 intracellular domain, a 4-1BB intracellular domain and a CD3ζ intracellular domain connected in sequence; the second-generation CAR includes a scFv sequence, a CD28 intracellular domain and a CD3ζ intracellular domain connected in sequence; or, the second-generation CAR includes a scFv sequence, a 4-1BB intracellular domain and a CD3ζ intracellular domain connected in sequence; the Foxp3 encoding gene is connected to the CD3ζ intracellular domain via a T2A peptide encoding gene.

[0007] The present invention also provides a CAR-T cell, comprising Foxp3 and a chimeric antigen receptor, wherein the amino acid sequence of the Foxp3 is shown in SEQ ID NO.2.

[0008] As an optional solution, the nucleotide sequence of the Foxp3 encoding gene is shown in SEQ ID NO.1.

[0009] The present invention also provides a method for preparing the CAR-T cells described in the above scheme, which are obtained by transfecting T lymphocytes with a recombinant lentivirus expressing Foxp3 and a chimeric antigen receptor.

[0010] The present invention also provides the use of the CAR-T cells described in the above scheme or the CAR-T cells prepared by the preparation method in the preparation of anti-tumor drugs.

[0011] As an alternative, the tumor comprises a hematological tumor or a solid tumor.

[0012] As an optional scheme, the solid tumor includes but is not limited to one or more of liver cancer, pancreatic cancer, ovarian cancer, bile duct cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer and breast cancer.

[0013] The present invention provides a fusion gene, the fusion gene includes a gene encoding Foxp3 and a gene encoding a chimeric antigen receptor (CAR); the amino acid sequence of the Foxp3 is shown in SEQ ID NO.2. Based on the fusion gene of the present invention, CAR-T cells that overexpress Foxp3 can be constructed, thereby simulating the metabolic pattern of regulatory T cells (Treg cells) and reshaping the metabolism of CAR-T cells to improve the anti-tumor effect. It has been verified that compared with CAR-T Conv Cells, CAR-T Foxp3 The cells have a stronger and longer-lasting anti-tumor effect and can exert long-term immune surveillance functions.

[0014] The present invention also provides a CAR-T cell, comprising Foxp3 and a chimeric antigen receptor, wherein the amino acid sequence of the Foxp3 is shown in SEQ ID NO.2. The CAR-T cell of the present invention is a Foxp3 metabolically regulated CAR-T cell. Compared with traditional second-generation or third-generation CAR-T cells, the tumor growth rate of the Foxp3 metabolically regulated CAR-T cells of the present invention is significantly slowed down, and it has a more powerful tumor suppression function, which can solve the problem of poor effect of CAR-T cell therapy due to the immunosuppressive microenvironment inside solid tumors. Mitochondria with a double membrane structure are always in a dynamic balance of fusion and fission, which is a necessary condition for maintaining T cell homeostasis, and also play an important role in the effector function and proliferation activation process of T cells. Mitochondrial fusion and fission are regulated by four mitochondrial power-related proteins. Mfn1, Mfn2 and Opa1 regulate mitochondrial fusion, while Drp1 controls mitochondrial fission. In the inactive state, Drp1 is usually located in the cytosol and phosphorylated at serine 637 to inhibit its activity. On the contrary, Drp1 activation requires phosphorylation of serine 616. During mitochondrial fission, Drp1 is recruited to the outer membrane of mitochondria and forms a ring structure around the mitochondria, causing the membrane to narrow. Then, GTP hydrolysis enhances this membrane contraction and promotes mitochondrial fragmentation. In the present invention, experimental techniques such as fluorescence bimolecular experiments, fluorescence co-localization and molecular docking have been used to confirm for the first time that the transcription factor Foxp3 can shuttle into the cytoplasm and interact with the mitochondrial power-related protein Drp1 to affect mitochondrial dynamics and then regulate cell metabolism, that is, Foxp3 regulates cell metabolism through mitochondria. Based on the above advantages, the present invention can provide the use of Foxp3 in the preparation of products that regulate CAR-T cell metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0016] Figure 1 Schematic diagram of the structure of GPC3-CAR and GPC3-CAR-Foxp3;

[0017] Figure 2 Flow cytometry for CAR-T Conv and CAR-T Foxp3 Cell positive rate result diagram;

[0018] Figure 3 This is the cell therapy flow chart in Experimental Example 1;

[0019] Figure 4 This is the result diagram of the tumor volume of mice in the T cell treatment group at the end point of the experiment in Experimental Example 1;

[0020] Figure 5 CAR-T is the end point of the experiment in Experiment 1 Conv Results of tumor volume of mice in the cell therapy group;

[0021] Figure 6 CAR-T is the end point of the experiment in Experiment 1 Foxp3 Results of tumor volume of mice in the cell therapy group;

[0022] Figure 7 This is a statistical graph of the tumor volume of each group of mice at the end of the experiment in Experimental Example 1;

[0023] Figure 8 This is the cell therapy flow chart in Experimental Example 2;

[0024] Fig. 9 This is the result diagram of the tumor volume of mice in the negative control group at the end of the experiment in Experimental Example 2;

[0025] Fig.10 CAR-T is the end point of the experiment in Experiment 2 Conv Results of tumor volume of mice in the cell therapy group;

[0026] Fig.11 CAR-T is the end point of the experiment in Experiment 2 Foxp3 Results of tumor volume of mice in the cell therapy group;

[0027] Fig.12 This is a statistical graph of the tumor volume of each group of mice at the end of the experiment in Experimental Example 2;

[0028] Fig.13 This is the result of spectral flow cytometry detection of different CAR-T cell surface markers;

[0029] Fig.14 This is a dimensionality reduction analysis diagram of different CAR-T cell surface markers detected by spectral flow cytometry;

[0030] Fig.15 Flow cytometry analysis of exhausted molecules (PD-1, TIM-3, LAG-3) after long-term antigen-specific stimulation of different CAR-T cells;

[0031] Fig.16 It indicates that Foxp3 and Drp1 colocalize;

[0032] Fig.17 This is a diagram showing the effect of Foxp3 on mitochondrial dynamics in CAR-T cells. DETAILED DESCRIPTION

[0033] The present invention provides a fusion gene, comprising a Foxp3 coding gene and a chimeric antigen receptor coding gene; the amino acid sequence of the Foxp3 is shown in SEQ ID NO.2.

[0034] In the present invention, the amino acid sequence shown in SEQ ID NO.2 is specifically:

[0035] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLK HCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEF LHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP.

[0036] As an optional solution, the nucleotide sequence of the Foxp3 coding gene is shown in SEQ ID NO.1, GenBank: EF534714.1, specifically:

[0037]

[0038] As an optional approach, the chimeric antigen receptor includes but is not limited to a third generation CAR or a second generation CAR.

[0039] As an optional embodiment, the second generation CAR comprises a scFv sequence, a 4-1BB intracellular domain (or a CD28 intracellular domain) and a CD3ζ intracellular domain connected sequentially.

[0040] As an optional scheme, the third generation CAR has dual co-stimulatory molecules, such as CD28 and 4-1BB; the second generation CAR has a single co-stimulatory molecule, such as CD28 or 4-1BB. As an optional embodiment, the third generation CAR includes a scFv sequence, a CD28 intracellular domain, a 4-1BB intracellular domain, and a CD3ζ intracellular domain connected in sequence.

[0041] As an optional embodiment, the Foxp3 encoding gene is connected to the CD3ζ intracellular domain via the T2A peptide encoding gene.

[0042] In the specific implementation of the present invention, any CAR can be selected; the CAR includes but is not limited to one or more of CD19-CAR, CD20-CAR, CD123-CAR, CD171-CAR, HER2 (human epidermal growth factor receptor 2) -CAR, GPC3 (Glypican 3) -CAR, BCMA (B cell maturation antigen) -CAR, MUC1 (Mucin 1, Cell Surface Associated) -CAR, PSMA (Prostate-specific membrane antigen) -CAR, NKG2D-CAR, GCC (guanylyl cyclase C) -CAR, MSLN (Mesothelin) -CAR and CEA (carcinoembryonic antigen) -CAR.

[0043] In the specific implementation of the present invention, the scFv sequence can target tumor antigens; depending on the type of cancer, the scFv sequence can be adjusted as needed. Taking liver cancer as an example, the amino acid sequence of the GPC3-scFv sequence is shown in SEQ ID NO.3, specifically:

[0044] MALPPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGASVKVSCKASGYTFSDYEMHWVRQAPGQGLEWMGAIHPGSGDTAYNQRFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARFYSYAYWGQGTLV TVSAGGGGSGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLQWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSIYVPYTFGQGTKLEIKR.

[0045] As an optional embodiment, the amino acid sequence of the CD28 intracellular domain is shown in SEQ ID NO.4, specifically:

[0046] FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.

[0047] As an optional embodiment, the amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID NO.5, specifically:

[0048] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0049] As an optional embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID NO.6, specifically:

[0050] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR.

[0051] As an optional embodiment, the amino acid sequence of the third-generation GPC3-CAR is shown in SEQ ID NO.7, specifically:

[0052] .

[0053] As an optional embodiment, the nucleotide sequence of the nucleic acid molecule encoding GPC3-CAR is shown in SEQ ID NO.8, specifically:

[0054]

[0055] As an optional embodiment, the nucleotide sequence of the nucleic acid molecule encoding GPC3-CAR-Foxp3 is shown in SEQ ID NO.9, specifically:

[0056]

[0057] As an optional embodiment, the present invention also provides a lentiviral expression vector, into which the fusion gene described in the above scheme is inserted. The present invention has no special requirements for the construction method of the lentiviral expression vector, and a method familiar to those skilled in the art can be used.

[0058] The present invention also provides a CAR-T cell, comprising Foxp3 and a chimeric antigen receptor, wherein the amino acid sequence of the Foxp3 and the nucleotide sequence of the encoding gene are as described above.

[0059] The CAR-T cells of the present invention are Foxp3 metabolically regulated CAR-T cells, which can significantly upregulate the expression of CD27, CD28 and CD62L, and significantly downregulate the expression of EOMES, PD-1, TIM-3 and LAG-3.

[0060] Compared with CAR-T Conv Cells, CAR-T Foxp3 The cells express higher levels of CD27 and CD28 molecules. After a long period of antigen-specific stimulation, CAR-T Foxp3 Cells still have a higher level of activation. Foxp3 The CD62L molecule level in the cell group was significantly higher than that in the CAR-T Conv CD62L, also known as L-selectin, is a cell adhesion molecule that is usually expressed in T cells or less differentiated T memory stem cells (Tscm) can promote the homing and migration of immune cells to the site of inflammation, while a higher proportion of T or Tscm cells have a stronger anti-tumor effect, and high expression of CD62L molecules confirms CAR-T Foxp3 Cells are in an earlier differentiation state, suggesting that CAR-T Foxp3 Cells have a more durable anti-tumor response. In addition, compared with CAR-T Conv Cells, CAR-T Foxp3 Cells express lower levels of EOMES. EOMES transcription factors have been shown to be involved in the process of T cell exhaustion. EOMES is highly expressed in exhausted CD8+T cells induced by chronic infection, and high levels of EOMES accelerate T cell exhaustion. Foxp3 Low expression of EOMES in cells also prompted CAR-T Foxp3 After long-term specific antigen stimulation, cells not only have a higher level of activation, but are also in a state of low exhaustion and early differentiation.

[0061] The present invention also provides a method for preparing the CAR-T cells described in the above scheme, which are obtained by transfecting T lymphocytes with a recombinant lentivirus expressing Foxp3 and a chimeric antigen receptor.

[0062] As an optional embodiment, the recombinant lentivirus is prepared by the following steps:

[0063] The lentiviral expression vector, psPAX2 and pMD2.G described in the above scheme were mixed and transfected into HEK293T cells. Fresh DMEM medium was replaced 8 to 12 hours after transfection, and the supernatant of HEK293T cells was collected for 36 hours and concentrated by ultracentrifugation to obtain recombinant lentivirus.

[0064] The present invention also provides the use of the CAR-T cells described in the above scheme or the CAR-T cells prepared by the preparation method in the preparation of anti-tumor drugs.

[0065] The present invention also provides the use of the CAR-T cells described in the above scheme in clinical tumor diseases.

[0066] The CAR-T cells (CAR-T Foxp3 ) can simulate the metabolic pattern of regulatory T cells, presenting a high activation and low exhaustion state in solid tumors, and at the same time has a stronger immune surveillance function. Compared with traditional CAR-T cells, it has a stronger anti-tumor ability.

[0067] As an alternative, the tumor comprises a hematological tumor or a solid tumor.

[0068] As an optional scheme, the tumor is a common tumor in clinical practice; the solid tumor includes but is not limited to one or more of liver cancer, pancreatic cancer, ovarian cancer, bile duct cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer and breast cancer.

[0069] The present invention provides a fusion gene, which includes a gene encoding Foxp3 and a gene encoding a chimeric antigen receptor (CAR). Based on the fusion gene of the present invention, CAR-T cells that overexpress Foxp3 can be constructed, thereby simulating the metabolic pattern of regulatory T cells (Treg cells) and reshaping the metabolism of CAR-T cells to improve the anti-tumor effect. It has been verified that compared with CAR-T Conv Cells, CAR-T Foxp3 The cells have a stronger and longer-lasting anti-tumor effect and can exert long-term immune surveillance functions.

[0070] In order to further illustrate the present invention, the CAR-T cells overexpressing Foxp3 and applications thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0071] Example 1

[0072] CAR-T cell preparation (taking GPC3-CAR as an example, see the structural diagram Figure 1 ):

[0073] (1) Design and construction of the third-generation GPC3-CAR sequence: extract the scFv sequence targeting the GPC3 antigen from the hu9F2 monoclonal antibody, and connect and combine it with the CD28 intracellular domain, the 4-1BB intracellular domain and the CD3ζ intracellular domain to form the third-generation GPC3-CAR, the amino acid sequence of which is shown in SEQ ID NO.7.

[0074] (2) Design and construction of the third-generation GPC3-CAR-Foxp3 sequence (see the schematic diagram for the structure) Figure 1 ): Based on the third-generation GPC3-CAR sequence, the human Foxp3 gene (GenBank: EF534714.1, the nucleotide sequence is shown in SEQID NO.1) is connected through the T2A peptide to achieve co-expression of the Foxp3 gene and the third-generation GPC3-CAR.

[0075] (3) Construction of lentiviral plasmids: The linearized lentiviral vector pWPT-GFP (Cat#12255, Addgene) was cut with the high-fidelity enzymes MluI and SalI, and the gene fragments encoding GPC3-CAR (nucleotide sequence as shown in SEQ ID NO.8) and GPC3-CAR-Foxp3 (nucleotide sequence as shown in SEQ ID NO.9) were cloned downstream of the promoter using homologous recombinases, respectively, to obtain GPC3-CAR lentiviral plasmids and GPC3-CAR-Foxp3 lentiviral plasmids.

[0076] (4) Lentivirus preparation: To produce lentivirus, the lentivirus packaging vector psPAX2 (Cat#12260, Addgene), pMD2.G (Cat#12259, Addgene) and CAR plasmid were transfected into HEK293T cells using polyethyleneimine (Polysciences, Warrington, USA). The CAR plasmid was GPC3-CAR lentiviral plasmid or GPC3-CAR-Foxp3 lentiviral plasmid. Fresh DMEM medium was replaced 8 to 12 hours after plasmid transfection. After 36 hours of co-transfection, the supernatant of HEK293T cells was collected for 36 hours, and the lentivirus was concentrated by ultracentrifugation to obtain lentivirus expressing GPC3-CAR and lentivirus expressing GPC3-CAR-Foxp3, respectively.

[0077] (5) CAR-T cell preparation: Healthy human PBMC was purified using the human T lymphocyte isolation kit Pan TCellIsolation Kit, human T lymphocytes were purified at 1*10 6 The cells were cultured at a density of 10 cells / mL in culture plates pre-coated with anti-CD3 (Biolegend) or anti-CD8 (Biolegend) antibodies. After 2 days of activation, T cells were activated with lentivirus expressing GPC3-CAR and lentivirus expressing GPC3-CAR-Foxp3, respectively, to prepare CAR-T cells. Conv Cells and CAR-T Foxp3 cells, and the positive rate was detected by flow cytometry. Figure 2 , the left picture shows CAR-T Conv Cells, the right picture is CAR-T Foxp3 Cells, it can be seen that CAR-T Foxp3 The cells successfully expressed CAR and Foxp3 genes.

[0078] Test Example 1

[0079] Animal Experimentation:

[0080] (1) Mouse preparation: One week before the start of the animal experiment, order 6-8 week old severely immunodeficient mice of uniform body weight and keep them in the animal room for one week to allow them to adapt to the new environment in advance.

[0081] (2) Subcutaneous tumor bearing: Huh7 cells, a liver cancer cell line, were revived and expanded. When the number of cells was sufficient for cell counting and tumor bearing, mice were subcutaneously bearing tumors on day 0. 2×10 6 Huh7 cells.

[0082] (3) Cell therapy: After 8 days of subcutaneous tumor implantation, the mouse tumor volume grew to 100 mm 3 After about 2-3 days, the mice were randomly divided into groups to ensure that the initial tumor volume was consistent among the groups. Conv Cell therapy group and CAR-T Foxp3 There were 5 mice in each cell therapy group. Each mouse in the T cell therapy group was injected with 1×10 6 T lymphocytes, CAR-T Conv Each mouse in the cell therapy group was injected with 1×10 6 CAR-T Conv Cells, CAR-T Foxp3 Each mouse in the cell therapy group was injected with 1×10 6 CAR-T Foxp3 Cells. See the flowchart for Figure 3 .

[0083] (4) Mouse monitoring: The growth and weight of the tumor were monitored throughout the experiment. 3 The monitoring end point.

[0084] The end point of the experiment was T cell therapy group and CAR-T Conv Cell therapy group and CAR-T Foxp3 The tumor volumes of mice in the cell therapy group are shown in Figures 4 to 6 The average results of each group can be found in Figure 7 , the end point of the experiment is CAR-T Foxp3 Cell Panel and CAR-T Conv The tumor volume after cell group peeling is shown in Table 1. It can be seen that the anti-tumor effect of CAR-T cells expressing Foxp3 gene was verified by in vivo experiments, in which T cells without any modification and traditional third-generation CAR-T cells were used as controls. The tumor volume and mouse weight changes were monitored throughout the treatment process. On the 20th day, it can be seen from the tumor growth curve that compared with CAR-T Conv Cell group, CAR-T Foxp3 The tumor growth curve of the cell group was slower, and the CAR-T Foxp3 The tumor volume and weight after cell peeling were smaller than those of CAR-T Conv Cell group.

[0085] Table 1 Experimental endpoints CAR-T Foxp3 Cell Panel and CAR-T Conv Tumor volume after cell group peeling

[0086] <![CDATA[CAR-T Conv ]]> <![CDATA[CAR-T Foxp3 ]]> <![CDATA[1683.749mm 3 ]]> <![CDATA[628.6044mm 3 ]]> <![CDATA[1050.715mm 3 ]]> <![CDATA[493.462mm 3 ]]> <![CDATA[1060.789mm 3 ]]> <![CDATA[1159.52mm 3 ]]> <![CDATA[1671.992mm 3 ]]> <![CDATA[1205.604mm 3 ]]> <![CDATA[1667.248mm 3 ]]> <![CDATA[362.0369mm 3 ]]>

[0087] Test Example 2

[0088] A tumor re-challenge model was constructed to verify the long-term anti-tumor response of CAR-T cells.

[0089] Severely immunodeficient mice were subcutaneously injected with tumors on day 0 (injection volume was 2×10 6 Huh7 cells), and on the 4th day, the tumor size was about 50 mm 3 At each time, 1×10 6 CAR-T Conv cells and 1×10 6 CAR-T Foxp3 cells were treated and injected again on day 29 with 8 × 10 6 Huh7 cells were used for re-tumoring. The flowchart is shown in Figure 8The mice of the same age without any treatment were used as negative control group. The negative control group consisted of 4 mice; Conv Cell experiment group, CAR-T injection Foxp3 There were 5 mice in each cell experiment group.

[0090] End point of the experiment: control group, CAR-T Conv Cell therapy group and CAR-T Foxp3 The tumor volumes of mice in the cell therapy group are shown in Fig. 9 , Fig.10 and Fig.11 The average results of each group can be found in Fig.12 , the end point of the experiment is CAR-T Foxp3 Cell Panel and CAR-T Conv The tumor volume of mice in the cell group is shown in Table 2. From the tumor growth curve, it can be seen that compared with CAR-T Conv Cell group, CAR-T Foxp3 The tumor growth rate of the cell group was significantly slowed down. These data once again confirmed that compared with CAR-T Conv Cells, CAR-T Foxp3 Cells have more powerful tumor suppressor functions.

[0091] Table 2 Experimental endpoints CAR-T Foxp3 Cell Panel and CAR-T Conv Tumor volume of mice in the cell group

[0092] <![CDATA[CAR-T Conv ]]> <![CDATA[CAR-T Foxp3 ]]> <![CDATA[1351.078mm 3 ]]> <![CDATA[588.9531mm 3 ]]> <![CDATA[923.3787mm 3 ]]> <![CDATA[249.0132mm 3 ]]> <![CDATA[1016.341mm 3 ]]> <![CDATA[370.1561mm 3 ]]> <![CDATA[786.8753mm 3 ]]> <![CDATA[276.5188mm 3 ]]> <![CDATA[976.0888mm 3 ]]> <![CDATA[315.7061mm 3 ]]> <![CDATA[780.2971mm 3 ]]> <![CDATA[226.9177mm 3 ]]>

[0093] Test Example 3

[0094] Measuring the anti-tumor function of CAR-T cells should consider the activation markers, differentiation phenotypes and key transcription factors on the cell surface. Multiple indicators are used in spectral flow cytometry to further explore the role of CAR-T cells in tumorigenesis. Foxp3 and CAR-T Conv Among them, CD27, CD28, and CD69 are used to evaluate CAR-T cell activation; CD45RA, CD62L, CD95, and CCR7 are used to identify the differentiation state of CAR-T cells; and EOMES and TCF-1 are transcription factors that play a key role in T cell differentiation and exhaustion.

[0095] The test process is as follows:

[0096] (1) Cell staining: CAR-T cells were collected and washed three days after GPC3 antigen-specific stimulation, and pretreated with Fc receptor blocking solution (Human TruStain FcX; Biolegend) before spectral flow cytometry antibody staining. Then, cells were stained with specific spectral flow cytometry antibodies and cell live-death dyes at 4°C for 30 minutes. After two rounds of washing, cells were resuspended and prepared for loading.

[0097] (2) Flow cytometry: After cell staining, the cells were analyzed using the spectral flow cytometer Cytek Aurora. At least 10,000 cells were collected for each sample, and unstained and untreated cells were used as negative controls.

[0098] (3) Data analysis: Flow cytometry raw data were analyzed using FlowJo software and t-Distributed Stochastic Neighbor Embedding (t-SNE) dimensionality reduction.

[0099] After streaming analysis of data ( Fig.13 ) and further t-SNE dimensionality reduction analysis ( Fig.14 ), it can be seen that compared with CAR-T Conv Cells, CAR-T Foxp3 The cells expressed higher levels of CD27 and CD28 molecules, which directly confirmed that after long-term antigen-specific stimulation, CAR-T Foxp3 Cells still have a higher level of activation. At the same time, it was also found that CAR-T Foxp3 The CD62L molecule level in the cell group was significantly higher than that in the CAR-T Conv CD62L, also known as L-selectin, is a cell adhesion molecule usually expressed in T cells or less differentiated T memory stem cells (Tscm) can promote the homing and migration of immune cells to the site of inflammation, while a higher proportion of T or Tscm cells have a stronger anti-tumor effect, and high expression of CD62L molecules confirms CAR-T Foxp3 Cells are in an earlier differentiation state, suggesting that CAR-T Foxp3 Cells have a more durable anti-tumor response. In addition, spectral flow cytometry data also showed that compared with CAR-T Conv Cells, CAR-T Foxp3The cells expressed lower levels of EOMES, while TCF-1 levels did not change significantly. EOMES transcription factors have been shown to be involved in the process of T cell exhaustion. EOMES is highly expressed in exhausted CD8+T cells induced by chronic infection, and high levels of EOMES accelerate T cell exhaustion. Foxp3 Low expression of EOMES in cells also prompted CAR-T Foxp3 After long-term specific antigen stimulation, cells not only have a higher level of activation, but are also in a state of low exhaustion and early differentiation.

[0100] In addition, CAR-T was detected by spectral flow cytometry. Foxp3 Cells have lower levels of EOMES, suggesting that CAR-T Foxp3 To address this problem, GPC3 antigen peptides were used to target different CAR-T cells (CAR-T Conv Cells and CAR-T Foxp3 After three days of specific stimulation, different CAR-T cells were collected and stained, and the expression levels of cell surface PD-1, TIM-3, and LAG-3 exhaustion markers were analyzed by flow cytometry. Fig.15 ) can be seen in that compared with CAR-T Conv Cells, CAR-T Foxp3 The expression levels of three depletion molecules, PD-1, TIM-3, and LAG-3, were significantly downregulated in the cells.

[0101] Test Example 4

[0102] (1) Remove CAR-T from the incubator Conv Cells and CAR-T Foxp3 The culture medium was discarded and the cells were plated onto glass slides.

[0103] (2) Cell fixation: Place cells in paraformaldehyde for fixation.

[0104] (3) Antigen blocking: The slides were placed in DPBS to wash away OCT and the cell tissues were circled with a histochemical pen, followed by blocking with 5% BSA at room temperature for 1 h.

[0105] (4) Staining: Dilute the primary antibody and stain overnight at 4°C, rinse three times with PBS before staining with the secondary antibody. Dilute the secondary antibody and stain for 1 hour at room temperature, rinse three times with PBS, and then use diluted DAPI to stain the cell nuclei.

[0106] (5) Sealing and imaging: Add mounting to the tissue area and cover with a coverslip for sealing, then use a confocal microscope for imaging. Fig.16 and Fig.17 .

[0107] Fig.16 For CAR-T conv Cells and CAR-T Foxp3 Images of cells under a fluorescence microscope; Drp1: shows the Drp1 protein labeled with red fluorescence; Foxp3: shows the Foxp3 protein labeled with green fluorescence; DAPI: shows the cell nucleus labeled with blue fluorescence; Merge: merges the above three fluorescent markers together to show the overall structure of the cell. Fig.17 For CAR-T conv Cells and CAR-T Foxp3 Fluorescence microscope images of cells; each type of cell has three different staining markers, including DAPI: blue, used to mark the cell nucleus; Mitochondria (Tom20): pink, used to mark mitochondria; Merge: merge the staining of DAPI and mitochondria to show the overlap of cell nuclei and mitochondria; the first row is CAR-T conv Cells; the second line is CAR-T Foxp3 cell; Fig.17 CAR-T conv Cells and CAR-T Foxp3 Cells differ in the distribution of their nuclei and mitochondria.

[0108] In summary, from Fig.16 and Fig.17 It can be seen that through experimental techniques such as fluorescence bimolecule experiments, fluorescence co-localization and molecular docking, it was confirmed for the first time that the transcription factor Foxp3 can shuttle into the cytoplasm and interact with the mitochondrial power-related protein Drp1 to affect mitochondrial dynamics and then regulate cellular metabolism. This is the first report of Foxp3 regulating cellular metabolism through mitochondria.

[0109] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fusion gene, characterized in that: It includes a gene encoding Foxp3 and a gene encoding a chimeric antigen receptor; the amino acid sequence of the Foxp3 is shown in SEQ ID NO.

2.

2. The fusion gene according to claim 1, characterized in that The nucleotide sequence of the Foxp3 coding gene is shown in SEQ ID NO.

1.

3. The fusion gene according to claim 1, characterized in that The chimeric antigen receptor includes but is not limited to a third-generation CAR or a second-generation CAR; the third-generation CAR includes a scFv sequence, a CD28 intracellular domain, a 4-1BB intracellular domain and a CD3ζ intracellular domain connected in sequence; the second-generation CAR includes a scFv sequence, a CD28 intracellular domain and a CD3ζ intracellular domain connected in sequence; or, the second-generation CAR includes a scFv sequence, a 4-1BB intracellular domain and a CD3ζ intracellular domain connected in sequence; the Foxp3 encoding gene is connected to the CD3ζ intracellular domain via a T2A peptide encoding gene.

4. A CAR-T cell, characterized in that: It comprises Foxp3 and a chimeric antigen receptor, wherein the amino acid sequence of Foxp3 is shown as SEQ ID NO.

2.

5. The CAR-T cell according to claim 4, characterized in that The nucleotide sequence of the Foxp3 coding gene is shown in SEQ ID NO.

1.

6. The method for preparing CAR-T cells according to claim 4 or 5, characterized in that: Obtained by transfecting T lymphocytes with a recombinant lentivirus expressing Foxp3 and chimeric antigen receptor.

7. Use of the CAR-T cells according to claim 4 or 5 or the CAR-T cells prepared by the preparation method according to claim 6 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The tumor includes a hematological tumor or a solid tumor.

9. The use according to claim 8, characterized in that: The solid tumors include, but are not limited to, one or more of liver cancer, pancreatic cancer, ovarian cancer, bile duct cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer and breast cancer.

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