Method for enhancing anti-tumor efficacy of immunotherapy T cells

Abstract

The invention relates to a method for enhancing the anti-tumor efficacy of immunotherapy T cells. Specifically, the invention relates to a method for enhancing the anti-tumor efficacy of immunotherapy T cells, and the method comprises the step of introducing MAFG genes into the immunotherapy T cells to express MAFG proteins. On the other hand, the invention relates to an immunotherapy T cell, a pharmaceutical composition containing the immunotherapy T cell and application of the immunotherapy T cell in preparation of drugs for treating tumors.

Description

Technical Field

[0001] The present application relates to the field of tumor prevention and treatment, and more specifically, to a method for enhancing the anti-tumor efficacy of immunotherapy T cells. Background Art

[0002] Immune cell therapy is a type of immunotherapy that injects immune cells (mainly T cells) into patients to treat cancer. The main types include chimeric antigen receptor T cell (CAR-T) therapy, T cell receptor engineered T cell (TCR-engineered T cell, TCR-T) therapy, tumor infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor NK cell (CAR-NK) therapy.

[0003] CAR-T cells refer to T cells activated in vitro and modified with chimeric antigen receptor (CAR) genes, amplified and then infused back into the patient's body. Through the specific receptors expressed by T cells, they can target and identify tumor cells and show strong killing activity. Currently, many CAR-T cell therapies have been launched in China and abroad for the treatment of blood tumors such as leukemia, lymphoma and myeloma.

[0004] TCR-T therapy is to obtain specific TCRs for tumor cells, use genetic engineering technology to transform T cells in vitro, and then infuse them back into the body to treat tumors. Its preparation process is similar to CAR-T therapy. The biggest difference is that the target antigens recognized by CAR-T therapy are all cell surface proteins, while TCR-T therapy can recognize intracellular antigen fragments presented by histocompatibility complex (MHC) molecules. Therefore, TCR-T therapy has a wider range of targets.

[0005] At present, although various therapies including CAR-T and TCR-T have shown excellent therapeutic prospects, the immune cells used for modification are mostly terminally differentiated T cells, which are prone to exhaustion, disability and aging, and are difficult to survive in the body for a long time, resulting in the prepared immune cells being in a terminal exhaustion state, which greatly affects the clinical effect of adoptive cell therapy products. In addition, due to the complex microenvironment of solid tumors, it is difficult for modified immunotherapy cells to infiltrate into the tumor. Even if a small number of cells infiltrate into the tumor, the immunosuppressive environment inside the tumor will induce the modified immunotherapy cells to enter an exhaustion state and fail to play an anti-tumor role.

[0006] MAF bZIP transcription factor G (MAFG) protein belongs to the small Maf protein of the Maf family. It is a type of transcription factor containing a basic domain and a basic leucine zipper (b-ZIP), which is abundantly expressed in embryonic cells and stem cells. Summary of the invention

[0007] This application is proposed in order to enhance the tumor killing and infiltration efficacy of existing T cell therapy products.

[0008] The preliminary experimental results of our laboratory showed that MAFG plays an important role in stem-like memory T cells (T SCM ) and acts as a transcription factor to activate the expression of stemness-related genes TCF7, SOX2, and NANOG. Compared with other known memory cells, T cells that highly express MAFG SCM The cells have a higher proliferation capacity, can be maintained in the body for a long time, and can quickly produce a large number of cytokines under the stimulation of antigens. Given that immune cells with high expression of MAFG have significantly improved self-proliferation ability and in vivo survival ability, enhancing and increasing the expression ratio of MAFG protein in immune cells will hopefully make up for the shortcomings of current CAR-T cell therapy and TCR-T therapy.

[0009] This application prepares MAFG-CAR T cells and MAFG-TCR T cells by inserting the MAFG gene into the corresponding plasmids for preparing CAR-T (Con-CAR T) cells and TCR-T (Con-TCR T) cells, respectively, and infecting T cells by lentiviral transduction. Immunotherapy T cells overexpressing MAFG have stronger proliferation ability, show stronger persistence and killing ability both in vivo and in vitro, and have more tumor infiltration and longer survival time.

[0010] In a first aspect, the present application relates to a method for enhancing the anti-tumor efficacy of immunotherapy T cells, the method comprising the following steps:

[0011] introducing the MAFG gene into the immunotherapeutic T cells to express the MAFG protein;

[0012] Preferably, the MAFG protein has an amino acid sequence as shown in SEQ ID NO: 2:

[0013] MTTPNKGNKALKVKREPGENGTSLTDEELVTMSVRELNQHLRGLS

[0014] KEEIVQLKQRRRTLKNRGYAASCRVKRVTQKEELEKQKAELQQEV

[0015] EKLASENASMKLELDALRSKYEALQTFARTVARSPVAPARGPLAAGLGPLLVPGKVAATSVITIVKSKTDARS (SEQ ID NO: 2).

[0016] Those skilled in the art can introduce genes into cells through conventional procedures, including but not limited to lentiviral methods, electroporation methods, liposome transfection methods, and the like.

[0017] In a specific embodiment, the immunotherapy T cells referred to herein refer to T cells used in immunotherapy injected into patients for the treatment of cancer, including but not limited to CAR-T cells and TCR-T cells.

[0018] Specifically, the present application relates to a method for enhancing the anti-tumor efficacy of immunotherapy T cells, the method comprising the following steps:

[0019] i) a synthetic construct comprising a MAFG gene and a chimeric antigen receptor (CAR) gene or an engineered T cell receptor (TCR) gene,

[0020] Preferably, the MAFG gene has a nucleotide sequence as shown in SEQ ID NO: 1:

[0021] ATGACGACCCCCAATAAAGGAAACAAGGCCTTGAAGGTGAAGC

[0022] GGGAGCCGGGTGAGAATGGCACCAGCCTGACGGATGAGGAGCT

[0023] GGTGACCATGTCGGTGCGGGAGCTGAACCAGCACCTGCGGGGC

[0024] CTGTCCAAGGAGGAGATCGTCCAGCTGAAGCAGCCGCCGGCGCA

[0025] CGCTCAAGAACCGCGGCTACGCTGCCAGCTGCCGCGTGAAGCGG

[0026] GTGACGCAGAAGGAGGAGCTGGAGAAGCAGAAGGCGGAGCTGC

[0027] AGCAGGAGGTGGAGAAGCTGGCCTCAGAGAACGCCAGCATGAA

[0028] GCTGGAGCTCGACGCGCTGCGCTCCAAGTACGAGGCTCTGCAGA

[0029] CCTTCGCCCGGACGGTGGCCCGCAGCCCCGTGGCCGCCAGCCCGG

[0030] GGCCCCTTTGCCGCCGGCCTGGGGCCCCTCGTCCCAGGCAAGGT

[0031] GGCCGCCACCAGCGTCATCACAATAGTAAAGTCCAAGACGGATGCCCGATCGTAG (SEQ ID NO: 1); and

[0032] ii) introducing the construct synthesized in step i) into the isolated T cells;

[0033] Preferably, the isolated T cells are derived from mammalian peripheral blood or tumors.

[0034] More preferably, the isolated T cells are derived from human peripheral blood or tumors.

[0035] As used herein, the isolated T cells are derived from mammalian peripheral blood or tumors, and do not mean that the method of the present application includes a step of obtaining peripheral blood or tumors from mammals. In fact, the isolated T cells used in the method of the present application can be obtained from isolated cultured T cells, or can be obtained commercially.

[0036] In a specific embodiment, the construct synthesized in step i) can be introduced into the isolated T cells by lentiviral, electroporation or liposome transfection.

[0037] In another aspect, the present application relates to an immunotherapy T cell comprising:

[0038] a) MAFG protein, preferably, the MAFG protein has an amino acid sequence as shown in SEQ ID NO: 2, and

[0039] b) Chimeric antigen receptor (CAR) or engineered T cell receptor (TCR).

[0040] In a specific embodiment, the MAFG protein is overexpressed in the immunotherapeutic T cells.

[0041] As used herein, "overexpression in immunotherapeutic T cells" means that after exogenously introducing the MAFG gene or protein, the expression level of the MAFG protein in the immunotherapeutic T cells is 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 times, or more of the expression level of the MAFG protein in the immunotherapeutic T cells without exogenously introducing the MAFG gene or protein.

[0042] In a specific embodiment, the immunotherapy T cells of the present application comprise a CAR or TCR targeting HER2. Thus, the immunotherapy T cells of the present application can be used to treat HER2-related tumors, including but not limited to gastric cancer, lung cancer, colon cancer, rectal cancer and ovarian cancer.

[0043] In a specific embodiment, the present application relates to an immunotherapy T cell comprising:

[0044] a) MAFG protein, preferably, the MAFG protein has an amino acid sequence as shown in SEQ ID NO: 2, and

[0045] b) a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR) antigen (Her2); exemplarily, the chimeric antigen receptor (CAR) comprises an antigen-specific scFv, a human CD8 signal peptide, a human CD8 hinge and transmembrane domain, a CD28 and 4-1BB co-stimulatory domain, and a CD3ζ intracellular domain.

[0046] Specifically, the antigen-specific scFv described herein is a specific scFv targeting Her2.

[0047] Compared with cells not containing MAFG protein, the immunotherapy T cells of the present application have one or more of the following characteristics:

[0048] 1) Enhanced in vitro proliferation capacity;

[0049] 2) Enhanced in vitro tumor cell killing ability;

[0050] 3) Enhanced in vitro cytokine release capacity;

[0051] 4) Enhanced tumor infiltration and cytokine release in vivo;

[0052] 5) Enhanced anti-tumor ability in vivo.

[0053] In another aspect, the present application relates to a pharmaceutical composition comprising the immunotherapeutic T cells of the present application and one or more pharmaceutically acceptable carriers.

[0054] In another aspect, the present application relates to the use of the immunotherapy T cells or pharmaceutical compositions of the present application in the preparation of a drug for treating a tumor in a subject.

[0055] Preferably, the tumor is selected from gastric cancer, lung cancer, colon cancer, rectal cancer, hematological tumors, prostate cancer, liver cancer, gallbladder cancer, kidney cancer, head and neck cancer, breast cancer and ovarian cancer, etc.

[0056] In some embodiments, the subject is a human or a non-human mammal, preferably the subject is a human.

[0057] In another aspect, the present application relates to the use of the MAFG gene or its encoded protein in the preparation of immunotherapeutic T cells, wherein:

[0058] allowing the MAFG gene or its encoded protein to be expressed in immunotherapy T cells;

[0059] Preferably, the nucleotide sequence of the MAFG gene is as shown in SEQ ID NO: 1, and

[0060] The amino acid sequence of the encoded protein is shown in SEQ ID NO: 2;

[0061] More preferably, the immunotherapy T cells are selected from CAR-T cells and TCR-T cells.

[0062] After a large number of experimental studies, the applicant found that compared with Con-CAR T and Con-TCR T cells, the modified T cells prepared by overexpressing MAFG protein have stronger expansion and killing capabilities in in vitro experiments. In addition, in vivo studies have shown that immune cells prepared by overexpressing this gene have stronger tumor infiltration ability and more persistent survival ability, thereby showing better anti-tumor effects. The therapeutic T cells and preparation methods of this application can significantly increase the tumor treatment efficacy of the corresponding cell therapy products, and have broad medical treatment prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1A and Figure 1C Schematic diagrams showing the plasmid vectors used to construct immunotherapy CAR-T cells and TCR-T cells that overexpress the MAFG gene, Figure 1B and Figure 1D The overexpression effect of MAFG was verified respectively.

[0064] Figure 2A and Figure 2BThe results showed that overexpression of MAFG gene could significantly enhance the proliferation ability of immunotherapy CAR-T cells and TCR-T cells.

[0065] Figure 3A and Figure 3B The results showed that overexpression of the MAFG gene can make immunotherapy CAR-T cells and TCR-T cells have stronger tumor cell killing ability in in vitro experiments.

[0066] Figure 4A and Figure 4B The results showed that overexpression of the MAFG gene can make immunotherapy CAR-T cells and TCR-T cells have stronger cytokine release ability in in vitro experiments.

[0067] FIG. 5A to FIG. 5D The results showed that immunotherapy CAR-T cells and TCR-T cells prepared by overexpressing the MAFG gene had stronger tumor infiltration and cytokine release abilities in in vivo experiments.

[0068] FIG. 6A to FIG. 6D The results showed that immunotherapy CAR-T cells and TCR-T cells prepared by overexpressing the MAFG gene had stronger anti-tumor ability in in vivo experiments. DETAILED DESCRIPTION

[0069] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and are not used to limit the present application.

[0070] Various cell lines and animals used in the examples:

[0071] Human peripheral blood was obtained from volunteers; human colorectal cancer cell line HCT116 was purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences; and immunodeficient NSG mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0072] Unless otherwise specified, the raw materials and reagents used in this application are commercially available.

[0073] Example 1: Successful overexpression of the MAFG gene in immunotherapy T cells.

[0074] 1. Experimental Procedure

[0075] 1.1 Using molecular cloning technology, construct MAFG-CAR T and MAFG-TCR T plasmids overexpressing the MAFG gene (SEQ ID NO: 1), and prepare corresponding lentiviruses;

[0076] Specifically, the preparation of lentivirus expressing CAR is as follows:

[0077] There are four types of lentiviral plasmids used in this experiment: Con-CAR T plasmid overexpressing human Her2 and MAFG-CAR T plasmid overexpressing MAFG on this basis, Con-TCR T plasmid overexpressing human Her2 and MAFG-TCR T plasmid overexpressing MAFG on this basis. The lentiviral packaging plasmids used in this experiment are pSPAX2 and PMD2G. The 293T cell lines were transfected with the above three target plasmids and pSPAX2 and PMD2G packaging plasmids to obtain lentiviruses expressing Con-CAR, MAFG-CAR, Con-TCR, and MAFG-TCR, respectively.

[0078] Taking the Con-CAR plasmid as an example, specifically, the plasmid is produced by fusing the human Her2 gene fragment into a lentiviral backbone plasmid containing an EF1a promoter. The plasmid consists of an antigen (Her2) specific scFv, a human CD8 signal peptide, a human CD8 hinge and transmembrane domain, CD28 and 4-1BB co-stimulatory domains, and a CD3ζ intracellular domain. Taking the MAFG-CAR T plasmid as an example, specifically, the plasmid is produced by overexpressing the human MAFG gene fragment on the basis of the Con-CAR plasmid and connecting it with T2A. The specific preparation steps of the lentivirus are as follows:

[0079] 1). Culture medium preparation

[0080] 293T cell complete culture medium: DMEM basal culture medium + 10% FBS + 33×Glutamax + 100× double antibody;

[0081] Virus packaging medium (prepared and used immediately): 293T cell complete medium + 500× sodium butyrate.

[0082] 2). Plasmid transfection

[0083] 293T cells were plated in a culture dish in advance, and plasmid transfection was performed when the cells grew to a density of 80-90%.

[0084] Preparation of transfection system for 10 cm culture dish: Add 250 μl Jet buffer to two 1.5 ml EP tubes respectively, gently add 40 μl Jet liposomes to one tube and gently mix, add target plasmid: PSPAX2:PMD2G=10 μg:7 μg:3 μg to the other tube and mix.

[0085] 3) Virus Concentration

[0086] Pretreatment of virus centrifugal column (30 kD, Merck): add 6 ml PBS to rinse the column, centrifuge at 5000 g for 5-7 min, and discard the liquid.

[0087] After 48 hours of changing the medium of the transfected 293T cells, collect the supernatant of the 10cm dish into a 50ml centrifuge tube, centrifuge at 1000g for 5min, and use a 20ml syringe to draw the supernatant after centrifugation, and filter it through a 0.45μm filter membrane. Add 15ml of the filtrate containing the virus after filtration to the pretreated virus concentration column, centrifuge at 5000g for 25min at 4℃, collect the virus concentrate on the concentration column and store it at -80℃. The titer of the concentrated virus is greater than 5×10 7 TU / ml, usually 1×10 8 TU / ml-5×10 8 TU / ml.

[0088] 1.2 Isolate and collect T cells from peripheral blood for the preparation of CAR-T cells; isolate and collect T cells from tumor cells for the preparation of TCR-T cells;

[0089] 1.3 Two types of T cells were cultured at an initial density of 1×10 6 100 μg / well were seeded into 24-well plates, and T cells were activated with CD3 (2.5 μg / ml), CD28 (1 μg / ml) monoclonal antibodies and cytokine IL-2 (100 IU / ml), and cultured in an incubator for 36 h;

[0090] 1.3.1 Collect T cells isolated and activated from peripheral blood and culture them into 1×10 6 CD8 + T cells were added with 100 μl of Con-CAR T concentrated virus or MAFG-CAR T concentrated virus without overexpressing MAFG, respectively, centrifuged at 1200 g for 2 h, and cultured in 24-well plates to prepare Con-CAR T cells and MAFG-CAR T cells overexpressing the MAFG gene.

[0091] 1.3.2 Collect T cells isolated and activated from tumor cells and culture them into 1×10 6 CD8 + T cells were added with 100 μl of Con-TCR T concentrated virus or MAFG-TCR T concentrated virus that did not overexpress MAFG, respectively, centrifuged at 1200 g for 2 hours, and cultured in a 24-well plate to prepare Con-TCR T cells and MAFG-TCR T cells that overexpressed the MAFG gene.

[0092] 1.4 After 3 days of culture, the immunotherapy CAR-T cells and TCR-T cells overexpressing MAFG and the corresponding control cells were collected, and the mRNA expression level of the MAFG gene was detected by qPCR.

[0093] 2. Experimental results

[0094] Figure 1A and Figure 1C Schematic diagrams of CAR-T and TCR-T plasmids overexpressing MAFG are shown respectively; qPCR results showed that MAFG was expressed in MAFG-CAR T cells ( Figure 1B ) and MAFG-TCR T cells ( Figure 1D ) was successfully overexpressed (p<0.0001).

[0095] Example 2: Immunotherapeutic T cells prepared by overexpressing the MAFG gene have stronger proliferation ability.

[0096] 1. Experimental Procedure

[0097] 1.1 After 3 days of in vitro culture, the MAFG gene-overexpressing CAR-T cells and corresponding controls were collected, and the total number of Con-CAR T cells and MAFG-CAR T cells was counted under a cell counting microscope.

[0098] 1.2 After 3 days of in vitro culture, the TCR-T cells overexpressing the MAFG gene and the corresponding controls were collected, and the total number of Con-TCR T cells and MAFG-TCR T cells were counted under a cell counting microscope.

[0099] 2. Experimental results

[0100] 2.1 Overexpression of MAFG can significantly increase the proliferation rate of CAR-T cells: Con-CAR T cells increased from the initial 5×10 6 amplified to 2.24×10 7 The number of T cells overexpressing MAFG-CAR was 3.86×10 7 significantly more than the control group (p<0.001) ( Figure 2A ).

[0101] 2.2 Overexpression of MAFG can significantly increase the proliferation rate of TCR-T cells: Con-TCR T cells increased from the initial 5×10 6 amplified to 1.01×10 7 The number of T cells overexpressing MAFG-TCR increased to 1.77×10 7 significantly more than the control group (p<0.001) ( Figure 2B ).

[0102] Example 3: Immunotherapy T cells prepared by overexpressing the MAFG gene have stronger tumor cell killing ability in in vitro experiments.

[0103] 1. Experimental Procedure

[0104] One day in advance, HCT116 tumor cells were cultured at a density of 5 × 10 4 The cells were plated in 24-well plates, and the MAFG-CAR T cells and MAFG-TCR T cells overexpressing the MAFG gene, as well as the Con-CAR T cells and Con-TCR T cells not overexpressing MAFG (both Her2 + T cells), flow cytometry sorting CAR + T cells were co-cultured with tumor cells at a 1:1 effector-target ratio. Two days later, the tumor cell lysis rate was detected and the suspended cells in the supernatant were collected by centrifugation. + T cells were co-cultured with tumor cells at the same effector-target ratio for two days for the second round of killing. This process was repeated for a total of 7 rounds of killing. The killing efficiency of CAR-T cells or TCR-T cells on tumor cells was tested in each round.

[0105] 2. Experimental results

[0106] The results showed that the killing efficiency of MAFG-CAR T cells and MAFG-TCR T cells prepared by overexpressing the MAFG gene against tumor cells in the first to seventh rounds was significantly higher than that of Con-CAR T cells and Con-TCRT cells that did not overexpress MAFG ( Figure 3A , Figure 3B ), showing a stronger ability to kill tumor cells in vitro.

[0107] Example 4: Immunotherapeutic T cells prepared by overexpressing the MAFG gene have stronger cytokine release ability in in vitro experiments.

[0108] 1. Experimental Procedure

[0109] One day in advance, HCT116 tumor cells were cultured at a density of 5 × 10 4 The cells were plated in 24-well plates, and the MAFG-CAR T cells and MAFG-TCR T cells overexpressing the MAFG gene, as well as the Con-CAR T cells and Con-TCR T cells not overexpressing MAFG (both Her2 + T cells), flow cytometry sorting CAR + T cells were co-cultured with tumor cells at a 1:1 effector-target ratio. Two days later, the suspended cells in the supernatant were collected by centrifugation. +T cells were co-cultured with tumor cells at the same effector-target ratio for two days for the second round of killing. The supernatant of cells after the second round of killing and co-incubation was collected by centrifugation, stained with antibodies such as IFN-γ, TNF-α, and IL-2 for 30 minutes, and data were collected using Invitrogen Attune NxT software. FlowJo software was used to analyze the data and detect the level of cytokine release in the co-culture.

[0110] 2. Experimental results

[0111] 2.1 Flow cytometry analysis of the cytokine release levels of Con-CAR T and MAFG-CAR T cells co-cultured with tumor cells revealed that the production of IFN-γ, TNF-α, and IL-2 by MAFG-CAR T cells was significantly increased (p<0.001; p<0.001; p<0.01)( Figure 4A ).

[0112] 2.2 Flow cytometry analysis of the cytokine release levels of Con-TCR T and MAFG-TCR T cells co-cultured with tumor cells revealed that the production of IFN-γ, TNF-α, and IL-2 by MAFG-TCR T cells was significantly increased (p<0.001; p<0.001; p<0.01)( Figure 4B ).

[0113] Example 5: Immunotherapy T cells prepared by overexpressing the MAFG gene have stronger tumor infiltration and cytokine release capabilities in in vivo experiments.

[0114] 1. Experimental Procedure

[0115] 1×10 6 HCT116 cells were inoculated into NSG mice. When the tumor grew to about 5×5 mm in size, 1×10 6 MAFG-CAR T cells, MAFG-TCR T cells overexpressing MAFG gene, and Con-CAR T cells and Con-TCR T cells not overexpressing MAFG (all Her2 + The number of immunotherapy T cells infiltrating into the tumor and the level of IFN-γ released by the immunotherapy T cells infiltrating into the tumor were detected on days 2, 7, and 14, respectively, to evaluate the infiltration ability of immunotherapy T cells in each group on solid tumors and the level of cytokine release in vivo.

[0116] 2. Experimental results

[0117] Flow cytometry analysis results showed that MAFG-CAR T cells and MAFG-TCR T cells prepared by overexpressing the MAFG gene could more easily infiltrate into tumor tissues ( Figure 5A , Figure 5C ) and had a higher IFN-γ release level ( Figure 5B , Figure 5D ).

[0118] Example 6: Immunotherapy T cells prepared by overexpressing the MAFG gene have stronger anti-tumor ability in in vivo experiments.

[0119] 1. Experimental Procedure

[0120] 1×10 6 HCT116 cells were inoculated into NSG mice. When the tumor grew to about 5×5 mm in size, 1×10 6 CAR + MAFG-CAR T cells and MAFG-TCR T cells that overexpress the MAFG gene, and Con-CAR T cells and Con-TCR T cells that do not overexpress MAFG (all Her2 + T cells), and the tumor size was measured every two days until the tumor grew to 2 cm 3 The animals were killed when the tumor volume and death time of tumor-bearing mice were recorded, and the tumor growth curve and survival period of tumor-bearing mice were drawn.

[0121] 2. Experimental results

[0122] The experimental results showed that compared with immunotherapy cells that did not overexpress the MAFG gene, MAFG-CAR T and MAFG-TCR T cells prepared by overexpressing the MAFG gene could significantly inhibit the growth of HCT116 tumors ( Fig. 6A , Figure 6C ) and prolonged the survival period of tumor-bearing mice ( Figure 6B , Fig.6D ).

[0123] In summary, the solution of this application has the following effects:

[0124] 1. The immunotherapy T cells prepared by overexpressing the MAFG gene in this application can significantly increase the proliferation rate of immunotherapy products.

[0125] 2. The immunotherapy T cells prepared by overexpressing the MAFG gene showed stronger tumor cell killing ability, cytokine release ability and infiltration ability in in vivo and in vitro studies, aiming to solve many problems of current immunotherapy cell therapy, such as short survival time in vivo and difficulty in infiltrating into the tumor, and have great clinical application prospects.

Claims

1. A method for enhancing the anti-tumor efficacy of immunotherapeutic T cells, the method comprising the following steps: introducing the MAFG gene into the immunotherapeutic T cells to express the MAFG protein; Preferably, the MAFG protein has an amino acid sequence as shown in SEQ ID NO:

2.

2. The method according to claim 1, wherein: The immunotherapy T cells are selected from CAR-T cells and TCR-T cells.

3. The method according to claim 1, comprising the steps of: i) a synthetic construct comprising a MAFG gene and a chimeric antigen receptor (CAR) gene or an engineered T cell receptor (TCR) gene, Preferably, the MAFG gene has a nucleotide sequence as shown in SEQ ID NO: 1; and ii) introducing the construct synthesized in step i) into the isolated T cells; Preferably, the isolated T cells are derived from mammalian peripheral blood or tumors. More preferably, the isolated T cells are derived from human peripheral blood or tumors.

4. The method according to claim 3, wherein: The construct synthesized in step i) is introduced into the isolated T cells via lentivirus.

5. An immunotherapy T cell comprising: a) MAFG protein, preferably, the MAFG protein has an amino acid sequence as shown in SEQ ID NO: 2, and b) Chimeric antigen receptor (CAR) or engineered T cell receptor (TCR), Preferably, the MAFG protein is overexpressed in the immunotherapeutic T cells.

6. The immunotherapeutic T cell according to claim 5, wherein the immunotherapeutic T cell has one or more of the following characteristics compared to a cell not comprising the MAFG protein: 1) Enhanced in vitro proliferation capacity; 2) Enhanced in vitro tumor cell killing ability; 3) Enhanced in vitro cytokine release capacity; 4) Enhanced tumor infiltration and cytokine release in vivo; 5) Enhanced anti-tumor ability in vivo.

7. A pharmaceutical composition comprising the immunotherapeutic T cells obtained according to any one of claims 1 to 4, or the immunotherapeutic T cells according to claim 5 or 6, and one or more pharmaceutically acceptable carriers.

8. Use of the immunotherapeutic T cells obtained according to any one of the methods of claims 1 to 4, or the immunotherapeutic T cells according to claim 5 or 6, or the pharmaceutical composition according to claim 7 in the preparation of a drug for treating a tumor in a subject, Preferably, the tumor is selected from gastric cancer, lung cancer, colon cancer, rectal cancer, hematological tumors, prostate cancer, liver cancer, gallbladder cancer, kidney cancer, head and neck cancer, breast cancer and ovarian cancer.

9. The use according to claim 8, wherein the subject is a human or a non-human mammal, preferably the subject is a human.

10. Use of the MAFG gene or its encoded protein in the preparation of immunotherapeutic T cells, wherein: allowing the MAFG gene or its encoded protein to be expressed in immunotherapy T cells; Preferably, the nucleotide sequence of the MAFG gene is as shown in SEQ ID NO: 1, and The amino acid sequence of the encoded protein is shown in SEQ ID NO: 2; Preferably, the immunotherapeutic T cells are selected from CAR-T cells and TCR-T cells.

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