Car-t cell overexpressing ifitm2 gene, construction method and application thereof in immune cell therapy

Abstract

The application provides a CAR-T cell overexpressing an IFITM2 gene, a construction method and application thereof in immune cell therapy, and relates to the technical field of biological medicine.The application takes CD276 as a target, regulates T cell memory differentiation and anti-exhaustion mechanism through overexpression of the IFITM2 gene, so that the treatment effect and persistence of CAR-T cell therapy are improved.The CD276-IFITM2 CAR-T cell constructed in the application has more memory differentiation phenotypes and fewer exhaustion phenotypes compared with the CD276 CAR-T cell, and through inhibition of PD-1 and LAG3, strong anti-exhaustion characteristics are realized.Meanwhile, the CD276-IFITM2 CAR-T cell significantly improves the expression level of IL-2, TNF alpha and IFN gamma under multiple antigen stimulations, and has the ability to continuously produce anti-cancer functional factors.In addition, the CD276-IFITM2 CAR-T cell has more significant tumor inhibition effect compared with the CD276 CAR-T cell.The CD276-IFITM2 CAR-T cell provided in the application can be used for preparing a drug for immune cell therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to CAR-T cells overexpressing the IFITM2 gene, their construction method, and their application in the preparation of immunotherapy drugs. Background Technology

[0002] CAR-T cell therapy is an adoptive immunotherapy method. It involves collecting the patient's own T cells and then genetically engineering them to express chimeric antigen receptors (CARs). CARs help T cells recognize specific antigens on the surface of tumor cells, enhancing their ability to kill tumor cells. After being cultured and expanded in vitro, the modified CAR-T cells are then reinfused into the patient to specifically attack tumor cells carrying the corresponding antigens. Currently, several CAR-T cell therapies have been approved for the treatment of hematological malignancies. For example, CD19-targeted CAR-T therapies have shown good efficacy in treating relapsed or refractory acute lymphoblastic leukemia and non-Hodgkin's lymphoma, with some patients achieving long-term complete remission; BCMA-targeted CAR-T therapies also show significant efficacy in treating multiple myeloma.

[0003] Currently, although CAR-T and other T-cell immunotherapies have achieved significant results in hematologic malignancies, they still face many challenges in the treatment of solid tumors. Among them, "T-cell exhaustion" is one of the key factors limiting its efficacy. Exhausted T cells gradually lose their killing function, proliferative capacity, and survival ability in the tumor microenvironment, manifested as high expression of immunosuppressive receptors and a reduction in effector factors. This functional failure not only leads to short-lasting efficacy but may also promote tumor immune escape. Therefore, how to overcome T-cell exhaustion and improve its functional sustainability has become an important scientific problem that CAR-T, TCR-T, and other immunocellular therapies urgently need to solve, and is of great significance for expanding their application in broader indications such as solid tumors. IFITM2 is an interferon-induced protein that is highly expressed in peripheral blood CD8+ T cells of very long-lived elderly individuals and is known to participate in antiviral immunity, but its role in T-cell metabolism and stemness maintenance remains unclear. This invention constructs CAR-T cells that express antigen receptor peptides and overexpress IFITM2, and studies their effects in tumor immunotherapy, providing a new treatment approach for CAR-T cell therapy in the treatment of solid tumors. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides CAR-T cells overexpressing the IFITM2 gene, a construction method thereof, and their application in immunotherapy.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a recombinant gene comprising the IFITM2 gene and a gene targeting a chimeric antigen receptor for CD276, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] Furthermore, the nucleotide sequence of the IFITM2 gene is shown in SEQ ID NO.2; the amino acid sequence of the protein encoded by the IFITM2 gene is shown in SEQ ID NO.3.

[0010] Furthermore, the nucleotide sequence of the gene targeting the chimeric antigen receptor for CD276 is shown in SEQ ID NO.4.

[0011] In a second aspect, the present invention provides a recombinant plasmid comprising a recombinant gene expressing a nucleotide sequence as shown in SEQ ID NO.1.

[0012] Thirdly, the present invention provides a recombinant lentivirus, which is prepared according to the following method: the recombinant plasmid, packaging plasmid psPAX2, and pMD2.G obtained in the second aspect are mixed and transfected into 293T cells. After transfection, the cells are cultured in DMEM complete medium for 6 hours. The cell supernatant is collected after 48 hours of transfection. After centrifugation, the supernatant is taken and filtered with a 0.45 μm filter. The filtrate is centrifuged at high speed and the supernatant is discarded, while the precipitate is retained to obtain the recombinant lentivirus.

[0013] Fourthly, this invention provides CAR-T cells overexpressing the IFITM2 gene, wherein the CAR-T cells are T lymphocytes transfected with the recombinant lentivirus obtained in the third aspect, and are specifically prepared according to the following steps:

[0014] (1) After activating the purified T cells for 48-50 hours, the above recombinant lentivirus was administered at a rate of 1×10⁻⁶. 6 Add 2 ml of recombinant lentiviral supernatant to each T cell, centrifuge at 3000 rpm and 32°C for 1.5 h, and replace with fresh culture medium after the centrifugation.

[0015] (2) Add the infected T cells to 1640 medium (containing 10% heat-inactivated FBS and 200 IU / ml IL-2), place them in an incubator, and replace the 1640 medium with fresh medium every 24 hours to obtain CAR-T cells that overexpress the IFITM2 gene, abbreviated as CD276-IFITM2 CAR-T cells.

[0016] Fifthly, the present invention provides the use of CAR-T cells overexpressing the IFITM2 gene in the preparation of drugs for immunocellular therapy.

[0017] (III) Beneficial Effects

[0018] This invention targets CD276 and regulates T cell memory differentiation and anti-exhaustion mechanisms by overexpressing the IFITM2 gene, effectively addressing the exhaustion problem of T cells in immunotherapy, enhancing their functional sustainability, and improving their therapeutic efficacy. Specifically, we constructed CD276-IFITM2 CAR-T cells that simultaneously express CD276 CAR and IFITM2 by adding the IFITM2 fragment to the CD276 CAR-T structure. Experimental results demonstrate that the CD276-IFITM2 CAR-T cells constructed in this invention exhibit more memory differentiation phenotypes and fewer exhaustion phenotypes compared to CD276 CAR-T cells, achieving strong anti-exhaustion characteristics by inhibiting PD-1 and LAG3. Simultaneously, CD276-IFITM2 CAR-T cells significantly increased the expression levels of IL-2, TNFα, and IFNγ under repeated antigen stimulation, demonstrating the ability to continuously produce anti-cancer functional factors. Furthermore, CD276-IFITM2 CAR-T cells exhibit a more significant tumor-suppressive effect compared to CD276 CAR-T cells. Attached Figure Description

[0019] Figure 1 The structures of CAR molecules in two types of cells, CD276 CAR-T and CD276-IFITM2 CAR-T, are shown.

[0020] Figure 2 The positive rates of CD276 CAR-T and CD276-IFITM2 CAR-T cells were measured.

[0021] Figure 3 Flow cytometry results for memory differentiation and exhaustion marker expression in CD276 CAR-T and CD276-IFITM2 CAR-T cells. CD276-IFITM2 CAR-T cells exhibited better memory differentiation and less exhaustion.

[0022] Figure 4 Flow cytometry results of intracellular functional factors in CD276 CAR-T and CD276-IFITM2 CAR-T cells 24 hours after each of three antigen stimulations. CD276-IFITM2 CAR-T cells exhibit a better ability to continuously produce functional factors.

[0023] Figure 5The expression of memory and exhaustion markers in CD276 CAR-T and CD276-IFITM2 CAR-T cells 7 days after antigen stimulation was shown. CD276-IFITM2 CAR-T cells exhibited better memory differentiation and less exhaustion.

[0024] Figure 6 This study examines the efficacy of CD276 CAR-T and CD276-IFITM2 CAR-T cells in an in vitro xenograft model. CD276-IFITM2 CAR-T showed better in vivo therapeutic efficacy. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0027] Example 1

[0028] Preparation of CD276-IFITM2 CAR-T cells

[0029] 1. Construction of lentiviral expression vector

[0030] 1.1 Construction of a lentiviral plasmid expressing a chimeric antigen receptor gene targeting CD276

[0031] The pCDH-SFFV-tNGFR-MSCV-CAR276(S33)-Flag-BBz(X2)-T2A plasmid was constructed using conventional genetic engineering techniques. The nucleotide sequence is shown in SEQ ID NO.4. The plasmid expresses a chimeric antigen receptor gene targeting CD276 (nucleotide sequence shown in SEQ ID NO.3).

[0032] 1.2 Construction of lentiviral plasmids expressing chimeric antigen receptor gene against CD276 and IFITM2 gene

[0033] (1) Based on the pCDH-SFFV-tNGFR-MSCV-CAR276(S33)-Flag-BBz(X2)-T2A plasmid, it was digested with SaII enzyme, and then the DNA fragment encoding IFITM2 (nucleotide sequence as shown in SEQ ID NO.2) was recombined into the pCDH-SFFV-tNGFR-MSCV-CAR276(S33)-Flag-BBz(X2)-T2A plasmid using homologous recombinase.

[0034] (2) The recombinant product from step (1) was transformed into Stb13 competent cells. Colonies were screened using plates containing ampicillin. Single clones were picked and cultured. Plasmids were extracted using a plasmid extraction kit. The recombinant lentiviral expression plasmids pCDH-SFFV-tNGFR-MSCV-CAR276(S33)-Flag-BBz(X2)-T2A and pCDH-SFFV-tNGFR-MSCV-CAR276(S33)-Flag-BBz(X2)-T2A-IFITM2-HA11 with correct sequencing results were preserved.

[0035] The nucleotide sequence of the pCDH-SFFV-tNGFR-MSCV-CAR276(S33)-Flag-BBz(X2)-T2A recombinant lentiviral expression plasmid is shown in SEQ ID NO.5.

[0036] The recombinant lentiviral plasmid pCDH-SFFV-tNGFR-MSCV-CAR276(S33)-Flag-BBz(X2)-T2A-IFITM2-HA11 expresses a recombinant gene consisting of the IFITM2 gene and a chimeric antigen receptor gene targeting CD276, the nucleotide sequence of which is shown in SEQ ID NO.1; the nucleotide sequence of the recombinant lentiviral expression plasmid is shown in SEQ ID NO.6.

[0037] 2. Preparation of recombinant lentivirus

[0038] Take one T75 culture flask containing 90% confluence 293T cells, collect the culture medium, wash twice with saline, add 0.8 mL of 0.05% trypsin, digest at 37°C for 3 min, add culture medium to stop digestion, pipette 10 μL for counting using a counting chamber, and then seed 1 × 10⁶ cells per well in a 6-well plate. 6293T cells were used in 6 wells. After 18 hours, the transfection medium was replaced with 2.5 mL. After 2 hours, the main plasmid (pCDH-SFFV-tNGFR-MSCV-CAR276(S33)-Flag-BBz(X2)-T2A-IFITM2-HA11) and packaging plasmids psPAX2 and pMD2.G were added to the wells using a calcium transfer kit. The plates were gently shaken and placed in an incubator. After 6 hours, the medium was replaced with 3 mL of complete medium. After 48 hours, the viral supernatant was collected to obtain recombinant lentivirus.

[0039] 3. Preparation of purified T cells

[0040] Peripheral blood from healthy individuals, saline, and lymphocyte separation medium were mixed in a 1:1:1 ratio and centrifuged at 2500 rpm for 25 minutes (increase speed 5, decrease speed 5). The white membrane layer was then aspirated and counted. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded. Based on the counting results, an appropriate amount of buffer and CD3 / CD28 sorting and activation magnetic beads were added. The cells were incubated at room temperature for 30 minutes. The incubated cell suspension was then added to the magnetic field, and after standing for 10 minutes, the waste liquid was discarded to obtain CD3+ T cells, which were then counted.

[0041] 4T cell infection

[0042] After activating the purified T cells for 48-50 hours, the recombinant lentivirus constructed above was administered at a rate of 1×10⁻⁶ cells / day. 6 Add 2 mL of viral supernatant to each T cell and centrifuge at 3000 rpm and 32°C for 1.5 h. Replace with fresh culture medium after centrifugation.

[0043] 5. Culture and expansion of CAR-T cells

[0044] Infected T cells were added to 1640 medium (containing 10% heat-inactivated FBS and 200 IU / mL IL-2), placed in an incubator, and the 1640 medium was replaced with fresh medium every 24 hours to obtain CAR-T cells overexpressing the IFITM2 gene, abbreviated as CD276-IFITM2 CAR-T cells.

[0045] Preparation of 6CD276 CAR-T cells

[0046] The only difference between the preparation methods of CD276 CAR-T cells and CD276-IFITM2 CAR-T cells is the lentiviral expression vector. Specifically, it is pCDH-SFFV-tNGFR-MSCV-CAR276(S33)-Flag-BBz(X2)-T2A plasmid. Any content not mentioned is completely the same as that of CD276-IFITM2 CAR-T cells.

[0047] Figure 1The structures of CAR molecules in two types of cells, CD276 CAR-T and CD276-IFITM2 CAR-T, are shown.

[0048] Expression efficiency of 7CAR molecules

[0049] After culturing CD276-IFITM2 CAR-T cells and CD276 CAR-T cells for 3 days, CAR T cells were labeled with Protein L from the APC channel, and the expression of gene editing on the surface of CAR T cells was detected by flow cytometry. Results are as follows: Figure 2 As shown, the expression efficiency of CAR molecules is 60% to 70%, indicating that the two types of CAR-T cells provided by this invention can be effectively prepared and can be used for subsequent experimental verification.

[0050] Example 2

[0051] Assay of CD276-IFITM2 CAR-T cell memory phenotype differentiation and exhaustion phenotype

[0052] Experimental method: CD276-IFITM2 CAR-T cells and CD276CAR-T cells were collected from the supernatant using 1.5ml EP tubes. After centrifugation at 500g for 5min, the supernatant was discarded, 1mL of flow buffer was added, vortexed, centrifuged at 500g for 5min, the supernatant was discarded, and 100μL of flow buffer was added to each tube.

[0053] Memory phenotypes: 1 μL FITC-anti-human NGFR, 1 μL BV785-Anti-human CD8, 1 μL PC5.5-anti-human CD4, 1 μL PE-cy7-anti-human CD62L, 1 μL APC-anti-human CD45ROA;

[0054] Exhaustion phenotype: 1 μL FITC-anti-human NGFR, 1 μL BV785-Anti-human CD8, 1 μL PE-cy7-anti-human CD4, 1 μL APC-anti-human CD366 (TIM3), 1 μL PC5.5-anti-human CD223 (LAG3)

[0055] After preparing the antibody suspension and mixing it well, add it to the cells, vortex and incubate at 4°C in the dark for 30 min. Add 1 mL of flow buffer, vortex and centrifuge at 500 g for 5 min. Add 200 μL of flow buffer and perform flow cytometry to detect CD62L, CD45RA, CD366 (TIM3), and CD223 (LAG3).

[0056] Experimental results

[0057] The results are as follows Figure 3 As shown, the proportions of CD45RA and CD62L double-positive cell populations in CD276-IFITM2 CAR-T cells and CD276 CAR-T cells were statistically analyzed. The positive proportions in the CD276 CAR-T cell group were 22.6, 22.7, and 22.4, while the positive proportions in the CD276-IFITM2 CAR-T cell group were 44.0, 45.4, and 44.3.

[0058] The proportions of CD223 and CD366 double-positive cells in the CD276-IFITM2 CAR T group and the CD276 CAR T group were statistically analyzed. The positive proportions in the CD276 CAR-T cell group were 19.6%, 18.7%, and 19.4%, respectively, while the positive proportions in the CD276-IFITM2 CAR-T cell group were 10.0%, 9.4%, and 10.3%, respectively.

[0059] The above results indicate that, under the same in vitro culture conditions, the CD276-IFITM2 CAR-T group exhibited more memory differentiation phenotypes and fewer exhaustion phenotypes compared to the CD276CAR-T group.

[0060] Example 3

[0061] The expression levels of IL-2, TNFα, and IFNγ were determined in the co-culture system.

[0062] Experimental methods: Aspc1 pancreatic cancer cell line was selected as the target cells. After counting, the cells were seeded into 12-well plates, with 1 × 10⁶ cells per well. 5 Cells were collected in triplicate, with CD276 CAR-T and CD276-IFITM2 CAR-T cells added at an effector-to-target ratio of 1:1. CD276 CAR-T cells served as a control. After 24 hours, the cells were counted, and new target cells were added at the same 1:1 ratio. This process was repeated three times. Before each count, CAR-T cells were collected from the supernatant in a 1.5 ml EP tube, centrifuged at 500 g for 5 min, and the supernatant was discarded. 1 ml of flow buffer was added, vortexed, and centrifuged at 500 g for 5 min. The supernatant was discarded, and 100 μl of PBS and 0.5 μl of eBioscience were added to each tube. TMFixableViability Dye eFluor TM Antibody suspensions were prepared using 450 live / dead dyes, 1 μL APC-anti-human CD45, 1 μL FITC-anti-human NGFR, 1 μL PC5.5-anti-human CD4, 1 μL BV785-anti-human CD8, 1 μL PEcy7-anti-human IL-2, 1 μL APCcy7-anti-human TNFα, and 1 μL AF700-anti-human IFNγ. After mixing, the suspensions were added to the cells, vortexed, and incubated at 4°C in the dark for 30 min. 1 mL of flow buffer was added, and the cells were vortexed again. After centrifugation at 500 g for 5 min, 200 μL of flow buffer was added. Relevant parameters were detected by flow cytometry, and the mean fluorescence intensity (MFI) of IL-2, TNFα, and IFNγ in CD276 CAR-T and CD276-IFITM2 CAR-T cells was calculated.

[0063] Experimental results:

[0064] The results are as follows Figure 4 As shown, the CD276-IFITM2 CAR-T group produced significantly more IL-2, TNFα, and IFNγ after repeated antigen stimulation compared to the CD276 CAR T group (P<0.05, P<0.001). This result indicates that after repeated antigen stimulation, CD276-IFITM2 CAR-T cells exhibit stronger anti-tumor activity compared to CD276 CAR-T cells.

[0065] Example 4

[0066] CD276-IFITM2 CAR-T cell exhaustion level test

[0067] Aspc1 pancreatic cancer cell line was selected as the target cells. After counting, the cells were seeded into 12-well plates, with 1 × 10⁶ cells per well. 5 Cells were collected in triplicate, and cultured for 12 hours. CD276-IFITM2 CAR-T and CD276CAR-T cells were added at a 1:1 effector-to-target ratio, with CD276CAR-T cells serving as a control. During the cell killing process, half the culture medium was replaced with fresh medium. After 7 days, CAR-T cells were collected from the supernatant in 1.5 ml EP tubes, centrifuged at 500 g for 5 min, and the supernatant was discarded. 1 ml of flow buffer was added, vortexed, and centrifuged at 500 g for 5 min, then the supernatant was discarded. CAR-T activation was assessed by characterizing cell exhaustion: 100 μL of PBS and 0.5 μL of eBioscience were added to each tube. TMFixableViability Dye eFluor TM Antibody suspensions were prepared using 450 μL of FITC-anti-human NGFR, 1 μL of BV785-Anti-human CD8, 1 μL of PE-cy7-anti-human CD4, 1 μL of PE-anti-human PD-1, and 1 μL of PE-anti-human CD223 (LAG3). After mixing, the suspensions were added to the cells, vortexed, and incubated at 4°C in the dark for 30 min. 1 mL of flow buffer was added, and the cells were vortexed again. After centrifugation at 500 g for 5 min, 200 μL of flow buffer was added. Relevant parameters were detected by flow cytometry. The mean fluorescence intensity (MFI) of PD-1 and LAG3 in the CD276 CAR T group and the CD276-IFITM2 CAR T group was calculated.

[0068] The results are as follows Figure 5 The results showed that the average fluorescence intensity of PD-1 and LAG3 in the CD276-IFITM2 CAR T group was significantly lower than that in the CD276 CAR T group (P<0.01, P<0.0001), indicating that the CD276-IFITM2 CAR T cells were significantly less depleted than the CD276 CAR T cells.

[0069] Example 5

[0070] CD276-IFITM2 CAR-T cell in vivo anti-tumor effect

[0071] Six-week-old NCG mice were subcutaneously inoculated with 5 × 10⁶ mice. 4 A xenograft model was constructed using the pancreatic cancer cell line Aspc1 (CD276 positive). After tumor formation was confirmed by in vivo imaging on day 5 of tumor bearing, the cells were randomly divided into three groups (n=5 / group): control group (UTD group, untreated), CD276 CAR-T cell group (1×10⁻⁶ cells / group), and control group (UTD group, untreated). 7 (cells / mouse) and CD276-IFITM2CAR-T cell group (1×10) 7 Cells (mice / mouse) were infused once via the tail vein on day 6 of tumor bearing, and tumor fluorescence values ​​were measured every 7 days thereafter to monitor tumor growth and survival.

[0072] The results are as follows Figure 6As shown, tumor fluorescence in the CD276-IFITM2 CAR T group was reduced by ≥85% and ≥60% compared to the UTD group and CD276CAR-T group, respectively, with significant differences (p<0.01). After 28 days, no tumor was observed in the CD276-IFITM2 CAR T group. These results indicate that CD276-IFITM2 CAR-T cells have a stronger anti-tumor effect than CD276 CAR-T cells.

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant gene, characterized in that The recombinant gene comprises an IFITM2 gene and a gene of a chimeric antigen receptor targeting CD276, and a nucleotide sequence of the recombinant gene is shown as SEQ ID NO.

1.

2. A recombinant gene according to claim 1, wherein, A nucleotide sequence of the IFITM2 gene is shown as SEQ ID NO. 2, and an amino acid sequence of a protein encoded by the IFITM2 gene is shown as SEQ ID NO.

3.

3. A recombinant gene according to claim 1, wherein, A nucleotide sequence of the gene of the chimeric antigen receptor targeting CD276 is shown as SEQ ID NO.

4.

4. A recombinant plasmid, characterized by The recombinant plasmid comprises a recombinant gene with an expression nucleotide sequence shown as SEQ ID NO.

1.

5. A recombinant lentivirus, characterized in that, The recombinant lentivirus is prepared by mixing the recombinant plasmid of claim 4, a packaging plasmid psPAX2 and pMD2.G, and then transfecting into 293T cells, replacing with DMEM complete medium 6 hours after transfection, collecting cell supernatant 48 hours after transfection, taking supernatant after centrifugation and filtering with a 0.45 μm filter head, high-speed centrifugation of the filtrate and discarding the supernatant, and retaining the precipitate, thereby obtaining the recombinant lentivirus.

6. CAR-T cells overexpressing the IFITM2 gene, characterized in that, The CAR-T cell is a T lymphocyte transfected with the recombinant lentivirus of claim 5, and is prepared according to the following steps: (1) After the purified T cells are activated for 48-50 h, 2 ml of the recombinant lentivirus supernatant is added per 1 x 10 6 T cells, and centrifuged at 3000 rpm and 32°C for 1.5 h. After the centrifugation, fresh culture medium is replaced. (2) adding the infected T cells into 1640 medium containing 10% heat-inactivated FBS and 200 IU / ml IL-2, and placing into a culture box, and replacing with fresh 1640 medium every 24 hours, thereby obtaining a CAR-T cell overexpressing an IFITM2 gene, referred to as CD276-IFITM2 CAR-T cell.

7. Use of the CAR-T cell overexpressing an IFITM2 gene of claim 6 in the preparation of a drug for immune cell therapy.

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