Application of ASPL-TFE3 fusion gene in preparation of medicine for enhancing curative effect of immune checkpoint inhibitor

Abstract

The invention provides an application of an ASPL-TFE3 fusion gene in preparation of a medicine for enhancing the curative effect of an immune checkpoint inhibitor. The nucleotide sequence of the fusion gene is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The invention relates to the field of tumor immunotherapy, in particular to a method for regulating and controlling expression and activity of TFE3 through ASPL-TFE3 fusion protein to enhance tumor cell autophagy and immune cell permeation, and the tumor treatment effect of an immune checkpoint inhibitor can be improved.

Description

Technical Field

[0001] The present invention belongs to the field of tumor immunotherapy, and specifically relates to the use of the ASPL-TFE3 fusion gene in the preparation of drugs for enhancing the efficacy of immune checkpoint inhibitors. Background Art

[0002] Alveolar soft part sarcoma (ASPS) is a rare, highly malignant soft tissue tumor, accounting for 0.5%-1% of all soft tissue sarcomas. It primarily occurs in adolescents and young adults, with a 5-year survival rate of only 20%-46%. It is prone to lung and brain metastasis, is poorly responsive to conventional chemotherapy, and has a high recurrence rate after surgical resection. Its molecular hallmark is the unbalanced chromosomal translocation der(17)t(X;17)(p11.2;q25), which results in the formation of the ASPL-TFE3 fusion gene.

[0003] The ASPL-TFE3 fusion gene is not only a diagnostic marker but also provides a specific target for immunotherapy. In recent years, immune checkpoint inhibitors (ICIs) have demonstrated significant potential in the treatment of ASPS. Single-agent PD-1 / PD-L1 inhibitors (such as pembrolizumab and nivolumab) have an ORR of approximately 15%-30%, with some patients experiencing long-term benefits. In a phase II clinical trial, the combination of bemosubishi (a PD-L1 inhibitor) and anlotinib (an anti-angiogenic TKI) achieved an ORR of 79.3%, including a complete response (CR) rate of 10.3% and a significantly prolonged median progression-free survival (PFS).

[0004] Tumor cells have long been the primary research target for combating malignant tumors. Tumor formation is a progressive, multi-gene, multi-factor, multi-stage, and multi-step pathological process, associated with abnormal cell proliferation, differentiation, and apoptosis, as well as the activation of numerous oncogenes and the abnormal expression of tumor suppressor genes. Malignant tumors impose a significant economic and social burden on both developed and underdeveloped countries. There is an urgent need to identify effective targeted therapies, which have positive implications for tumor treatment.

[0005] Therefore, designing a new immunotherapy regimen based on the changes in tumor autophagy levels and tumor microenvironment regulation mechanisms mediated by the ASPL-TFE3 fusion gene has important application prospects in current tumor treatment. Summary of the Invention

[0006] To address the shortcomings of the existing technology, the present invention aims to provide the use of an ASPL-TFE3 fusion gene in the preparation of a drug that enhances the efficacy of immune checkpoint inhibitors. This novel strategy enhances the efficacy of PD-L1 antibody therapy by enhancing tumor cell autophagy and activating the immune response, thereby improving the clinical treatment of tumors such as alveolar soft tissue sarcoma.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an ASPL-TFE3 fusion gene, the nucleotide sequence of the fusion gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] The present invention relates to the field of tumor immunotherapy. The present invention uses an ASPL-TFE3 fusion protein to regulate TFE3 expression and activity to enhance tumor cell autophagy and immune cell infiltration, thereby improving the efficacy of immune checkpoint inhibitors in treating tumors.

[0010] Based on the changes in tumor autophagy levels and tumor microenvironment regulation mechanisms mediated by the ASPL-TFE3 fusion gene, the present invention designs a new immunotherapy regimen, which is expected to solve the clinical problems of high drug resistance and limited efficacy in current tumor treatment.

[0011] In a specific embodiment of the present invention, the present invention uses genetic engineering methods to express the ASPL-TFE3 fusion gene in MCA205 cells and TC-1 cells, and it is found that the ASPL-TFE3 fusion gene can significantly enhance the autophagy level of tumor cells and tumor tissues.

[0012] In a second aspect, the present invention provides an expression vector comprising the nucleotide sequence of the ASPL-TFE3 fusion gene described in the first aspect.

[0013] In a third aspect, the present invention provides a recombinant virus, wherein the recombinant virus carries the nucleotide sequence of the ASPL-TFE3 fusion gene described in the first aspect.

[0014] Preferably, the recombinant virus is a lentivirus.

[0015] In a fourth aspect, the present invention provides use of the ASPL-TFE3 fusion gene described in the first aspect in the preparation of a drug for enhancing the efficacy of an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a PD-L1 antibody.

[0016] Preferably, the ASPL-TFE3 fusion gene is introduced into tumor cells via a viral vector to express the ASPL-TFE3 fusion protein.

[0017] Preferably, after the tumor cells express the ASPL-TFE3 fusion protein, immune activation and anti-tumor effects are promoted.

[0018] Preferably, the ASPL-TFE3 fusion protein enhances the autophagy level of tumor cells.

[0019] Preferably, the PD-L1 treatment increases CD8 + T cell infiltration.

[0020] Preferably, the tumor is a fibrosarcoma or a lung epithelial tumor.

[0021] In one embodiment of the present invention, a tumor model expressing ASPL-TFE3 fusion protein was constructed, which was responsive to PD-L1 treatment. PD-L1 treatment increased CD8 + T cell infiltration; compared with wild-type TFE3 overexpression or wild-type tumor cell controls, the ASPL-TFE3 fusion protein promoted immune activation and anti-tumor effects. Based on this, the present invention provides a novel strategy for regulating TFE3 protein expression through the ASPL-TFE3 fusion gene, enhancing tumor cell autophagy and activating the immune response, thereby improving the efficacy of PD-L1 antibody therapy. This strategy has important application prospects in tumor treatment.

[0022] In a fifth aspect, the present invention provides a composition for treating tumors, comprising the ASPL-TFE3 fusion gene described in the first aspect, the expression vector described in the second aspect, or the recombinant virus described in the third aspect.

[0023] Preferably, the composition further comprises an immune checkpoint inhibitor.

[0024] Preferably, the composition further comprises a pharmaceutically or immunologically acceptable carrier, excipient or adjuvant.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) Expression of ASPL-TFE3 fusion gene in MCA205 and TC-1 cells can significantly enhance autophagy level. After PD-L1 treatment of tumor cells expressing ASPL-TFE3 fusion protein, CD8 + T cell infiltration; compared with the wild-type TFE3 overexpression or wild-type tumor cell control group, ASPL-TFE3 fusion protein has the effect of promoting immune activation and anti-tumor effects.

[0027] (2) This invention designs a new immunotherapy scheme based on the changes in tumor autophagy levels mediated by the ASPL-TFE3 fusion gene and the tumor microenvironment regulation mechanism, which is expected to solve the clinical problems of high drug resistance and limited efficacy in current tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This figure shows the effect of ASPL-TFE3 fusion gene on cell autophagy level and tumor growth.

[0029] Figure 2 The results of immunohistochemical staining.

[0030] Figure 3 This figure shows the results of observing the autophagy level in tumor tissues by staining LC3 through immunohistochemistry experiments.

[0031] Figure 4 The response of ASPL-TFE3 fusion gene overexpressing tumors to PD-L1 antibody treatment.

[0032] Figure 5 CD8 in ASPL-TFE3 fusion gene overexpressing tumors after PD-L1 antibody treatment + T cell infiltration. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0034] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0035] Example 1

[0036] Effects of ASPL-TFE3 fusion protein on autophagy and tumor growth

[0037] To explore the effects of ASPL-TFE3 fusion protein on autophagy and tumor growth, in this example, an ASPL-TFE3 fusion protein overexpressing cell line was constructed and mouse experiments were conducted to observe the effects of ASPL-TFE3 fusion protein on tumor growth.

[0038] 1. Use lentiviral technology to construct ASPL-TFE3 fusion gene and wild-type TFE3 overexpression cell lines

[0039] (1) Search for the protein coding region (CDS) you want to overexpress in NCBI, then add appropriate enzyme cleavage sites and submit it to Suzhou Jinweizhi Company for synthesis.

[0040] The gene sequence of wild-type TFE3 (SEQ ID NO: 5).

[0041]

[0042] Nucleotide sequence corresponding to ASPL-TFE3 fusion protein:

[0043] ASPL-TFE3 fusion gene type 1 (SEQ ID NO: 1).

[0044]

[0045] ASPL-TFE3 fusion gene type 2 (SEQ ID NO: 2).

[0046]

[0047] Protein sequence:

[0048] ASPL-TFE3 fusion protein type 1 (SEQ ID NO:3).

[0049] MAAPAGGGGSAVSVLAPNGRRHTVKVTPSTVLLQVLEDTCRRQDFNPCEYDLKFQRSVLDLSLQWRFANLPNNAKLEMVPASRSREGPENMVRIALQLDDGSRLQDSFCSGQTLWELLSHFPQIRECLQHPGGATPVCVYTRDEVTGEAALRGTTLQSLGLTGGSATIRFVMKCYDPVGKTPGSLGSSASAGQAAASAPLPLESGELSRGDLSRPEDADTSGPCCEHTQEKQSTRAPAAAPFVPFSGGGQRLGGPPGPTRPLTSSSAKLPKSLSSPGGPSKPKKSKSGQDPQQEQEQERERDPQQEQERERIDDVIDEIISLESSYNDEMLSYLPGGTTGLQLPSTLPVSGNLLDVYSSQGVATPAITVSNSCPAELPNIKREISETEAKALLKERQKKDNHNLIERRRRFNINDRIKELGTLIPKSSDPEMRWNKGTILKASVDYIRKLQKEQQRSKDLESRQRSLEQANRSLQLRIQELELQAQIHGLPVPPTPGLLSLATTSASDSLKPEQLDIEEEGRPGAATFHVGGGPAQNAPHQQPPAPPSDALLDLHFPSDHLGDLGDPFHLGLEDILMEEEEGVVGGLSGGALSPLRAASDPLLSSVSPAVSKASSRRSSFSMEEES

[0050] ASPL-TFE3 fusion protein type 2 (SEQ ID NO:4).

[0051] .

[0052] (2) Lentiviral packaging

[0053] Transfection was performed when the cell density reached about 60%-70%. The target plasmid and packaging plasmids psPAX2 and pMD2.G were mixed in a 4:3:1 ratio to form a 2 μg / well system, then diluted with 200 μL of OPTI, and then 8 μL of PEI was added, mixed, and allowed to stand for 15 minutes. After standing, the mixture was added to one well of a 6-well plate. After 4-6 hours, the cells in the 6-well plate were replaced with a medium. After 48 hours, the lentivirus was collected and filtered with a 0.45 μM filter membrane. The aliquots were frozen at -80°C or immediately concentrated and used to infect the target cells.

[0054] (3) The packaged lentivirus was used to infect the target cells. After 48 hours of infection, the fluorescently labeled cells were enriched using a flow cytometry instrument to obtain the target cells (MCA205-WT, MCA205-TFE3, MCA205-ASPL-TFE3 and TC-1-WT, TC-1-TFE3, TC-1-ASPL-TFE3).

[0055] (4) After the enriched positive cells have grown, protein immunoblotting is performed to verify whether the overexpression is successful at the protein level.

[0056] 2. Immunoblotting assay to detect autophagy levels

[0057] (1) Preparation of protein samples

[0058] Cell collection: Take out the cell culture dish, collect the cells, then wash the cell pellet with PBS and add an appropriate volume of lysis buffer.

[0059] Protein extraction: Place the cell pellet resuspended in lysate on ice for 30 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes. After centrifugation, remove a volume of the supernatant and place it in a new EP tube. Add 4x SDS loading buffer, mix thoroughly, and heat in a 100°C water bath for 10 minutes. Store the collected protein sample at -20°C or use immediately.

[0060] (2) Preparation of polyacrylamide gel

[0061] Prepare gel according to the instructions of Yazyme polyacrylamide gel

[0062] (3) Loading and running the gel

[0063] Add samples to the wells according to the designated sample sequence, flanking the wells with a 26616 protein molecular weight marker. After sample loading, initially run the gel at 80 V. Once the samples have passed through the top stacking gel, adjust the voltage to 120 V.

[0064] (4) Transfer

[0065] Activate the PVDF membrane with methanol. Remove the protein gel and cut off the stacking gel. Place the membrane in a transfer cassette in the order of sponge, filter paper, gel, PVDF membrane, filter paper, and sponge, removing any air bubbles. Finally, place the transfer cassette in a transfer tank with two ice cubes, add the transfer solution, and close the lid. Adjust the current to 320 mA and the timer to 2 hours to begin the transfer.

[0066] (5) Skim milk seal

[0067] After the transfer is completed, 5% skim milk is prepared with TBST as a blocking solution, and the PVDF membrane is placed in the blocking solution and blocked at room temperature for 1 hour.

[0068] (6) Primary antibody incubation

[0069] The primary antibody was prepared in universal antibody diluent according to the ratio recommended in the antibody manual. The PVDF membrane was cut according to the size of the target molecule and placed in the primary antibody solution and incubated with slow shaking at 4°C overnight.

[0070] (7) Wash the membrane: Wash rapidly with TBST three times, 15 minutes each time.

[0071] (8) Secondary antibody incubation: Secondary antibody (TBST preparation), incubate at room temperature for 1 hour.

[0072] (9) Wash the membrane: Wash rapidly with TBST three times, 15 minutes each time.

[0073] (10) Development: The luminescent mixture is infiltrated into the PVDF membrane, and then exposed using a fluorescent chemiluminescence imager.

[0074] 3. Mouse subcutaneous transplant tumor model

[0075] (1) Mouse husbandry and grouping

[0076] All animals were housed in a specific pathogen-free facility. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the Suzhou Institute of Systems Medicine. C57BL / 6N females (6–8 weeks old, weighing 18–20 g) were purchased from Zhejiang Weitong Lihua Co., Ltd. Mice were randomly divided into groups of five before inoculation.

[0077] (2) Cell treatment

[0078] The tumor cells to be loaded with tumors (MCA205-WT, MCA205-TFE3, MCA205-ASPL-TFE3 and TC-1-WT, TC-1-TFE3, TC-1-ASPL-TFE3) were digested and prepared into single-cell suspensions. The cells were washed twice with PBS and the cell concentration was adjusted to 2 × 10 6 The cells were resuspended in PBS solution and used for tumor loading.

[0079] (3) Tumor growth detection

[0080] Tumor cells were inoculated into the right side of the back of C57BL / 6N mice, with 2×10 cells inoculated per mouse. 6 Starting from day 7, the tumor size was measured every 2-3 days using a caliper and the tumor area was calculated by multiplying the length by the width. When the tumor area exceeded 300 mm 2That is, the mice were euthanized.

[0081] 4. Tumor tissue immunohistochemistry experiment

[0082] (1) Frozen section sample preparation

[0083] The tumor tissue was surgically isolated and immersed in 4% paraformaldehyde, fixed overnight at 4°C, and placed on a turntable with the turntable speed adjusted to medium.

[0084] The tumor tissue was then transferred to a 30% sucrose solution (prepared with 1× PBS) and dehydrated for 24-48 hours until the tumor tissue completely sank to the bottom. The tumor tissue was then picked up and dried, and embedded in OCT embedding medium and frozen at -20°C.

[0085] The embedded tumor tissue was sliced ​​using a platform tissue slicer to a thickness of about 5 μm and stored at -20°C for subsequent staining.

[0086] (2) Fluorescent staining

[0087] After the frozen tissue sections returned to room temperature, they were washed with PBS to remove the embedding medium.

[0088] Membrane permeabilization: Infiltrate the tissue with 0.1% Triton solution and let it stand for 10 minutes.

[0089] Block with blocking solution at room temperature for about 30 minutes (blocking solution: 10% FBS in PBS solution).

[0090] Primary antibody incubation: Prepare the primary antibody solution (Cell Signaling Technology, 83506) using PBS containing 10% FBS according to the ratio recommended in the antibody manual and incubate overnight at 4°C.

[0091] The primary antibody solution was recovered the next day, and the sections were washed three times with PBS containing 10% FBS, each time for about 5 minutes.

[0092] Secondary antibody incubation: Use PBS containing 10% FBS to prepare the secondary antibody solution (Cell Signaling Technology, 7076) according to the ratio recommended in the antibody manual and incubate at room temperature for 2 hours.

[0093] After the secondary antibody incubation is completed, the sections were washed three times with PBS, each time for about 5 minutes.

[0094] Staining of cell nuclei: Hochest (Thermo Fisher Scientific, H3569) dye (1:1000) was diluted in PBS and the sections were stained for 15 minutes.

[0095] Then wash three times with PBS, let it stand for about 5 minutes each time, add anti-quenching agent, seal the slide, and then take it to the platform microscope for photography.

[0096] 5. ASPL-TFE3 fusion protein tumor response to PD-L1

[0097] PD-L1 antibody therapy for tumors expressing ASPL-TFE3 fusion protein

[0098] (1) Construction of mouse model

[0099] Tumor cells (MCA205-WT, MCA205-TFE3, MCA205-ASPL-TFE3 and TC-1-WT, TC-1-TFE3, TC-1-ASPL-TFE3) were subcutaneously transplanted into the back of C57BL / 6N mice, with an inoculation rate of 2 × 10 cells per mouse. 6 Seven days after tumor loading, mice were injected with 200 μg of PD-L1 antibody via the tail vein, once every other day, for a total of three injections. A control group was treated with PBS. Starting on day 7, tumor size was measured with a caliper every 2-3 days.

[0100] From the above experiments, it can be seen that PD-L1 antibodies can effectively control the growth of tumors expressing ASPL-TFE3 fusion protein.

[0101] (2) Tumor tissue immunohistochemical CD8 staining experiment

[0102] Preparation of frozen section samples: Tumor tissue was surgically isolated and immersed in 4% paraformaldehyde, fixed overnight at 4°C, and placed on a turntable with the turntable speed set to medium.

[0103] The tumor tissue was then transferred to a 30% sucrose solution (prepared with 1× PBS) and dehydrated for 24-48 hours until the tumor tissue completely sank to the bottom. The tumor tissue was then picked up and dried, and embedded in OCT embedding medium and frozen at -20°C.

[0104] The embedded tumor tissue was sliced ​​using a platform tissue slicer to a thickness of about 5 μm and stored at -20°C for subsequent staining.

[0105] Fluorescent staining: After the frozen tissue sections were returned to room temperature, they were washed with PBS to remove the embedding medium.

[0106] Membrane permeabilization: Infiltrate the tissue with 0.1% Triton solution and let it stand for 10 minutes.

[0107] Block with blocking solution at room temperature for about 30 minutes (blocking solution: 10% FBS in PBS solution).

[0108] Primary antibody incubation: Prepare the primary antibody solution (Cell Signaling Technology, 98941) using PBS containing 10% FBS according to the ratio recommended in the antibody manual and incubate overnight at 4°C.

[0109] The primary antibody solution was recovered the next day, and the sections were washed three times with PBS containing 10% FBS, each time for about 5 minutes.

[0110] Secondary antibody incubation: Prepare secondary antibody solution (Cell Signaling Technology, 7076) using PBS containing 10% FBS according to the ratio recommended in the antibody manual and incubate at room temperature for 2 hours.

[0111] After the secondary antibody incubation is completed, the sections were washed three times with PBS, each time for about 5 minutes.

[0112] Staining of cell nuclei: Dilute Hochest dye (1:1000) in PBS and stain the sections for 15 minutes.

[0113] Then wash three times with PBS, let it stand for about 5 minutes each time, add anti-quenching agent, seal the slide, and then take it to the platform microscope for photography.

[0114] Figure 1 The results show that the ASPL-TFE3 fusion gene was overexpressed in wild-type MCA205 and TC-1 tumor cells using lentiviral technology to observe the effect of this fusion gene on cellular autophagy levels and tumor growth. Figure 1 Figures A and D in the middle illustrate that the ASPL-TFE3 fusion gene can increase the autophagy level of tumor cells; Figure 1 Figures BC and EF in the middle illustrate that the ASPL-TFE3 fusion gene can promote tumor growth.

[0115] Figure 1 In Figures A and D, "1" represents "WT", "2" represents "TFE3", "3" represents "fusion protein 1", and "4" represents "fusion protein 2".

[0116] Figure 2 This is the result of immunohistochemical staining. It can be seen from the figure that ASPL-TFE3 fusion gene can increase the autophagy level of tumor tissue.

[0117] Figure 3 To observe the statistical results of autophagy levels in tumor tissues by immunohistochemical staining of LC3, Figure 3A in the middle is a statistical diagram of LC3 staining scores of overexpressed ASPL-TFE3 fusion gene, wild-type TFE3 and wild-type MCA205 tumor tissues. Figure 3 As shown in Figure A, overexpression of the ASPL-TFE3 fusion gene can increase the level of autophagy in tumor tissue. Figure 3 Middle B is the LC3 staining score statistical diagram of overexpressed ASPL-TFE3 fusion gene, wild-type TFE3 and wild-type TC-1 tumor tissues. Figure 3 As shown in Figure B, overexpression of the ASPL-TFE3 fusion gene can increase the level of autophagy in tumor tissue. Figure 3 The results showed that the autophagy level of tumor tissue overexpressing ASPL-TFE3 fusion gene was higher than that of the control group.

[0118] Figure 4 The results show the response of ASPL-TFE3 fusion gene-overexpressing tumors to PD-L1 antibody treatment; Figure 4 A in the middle is the growth curve of the response of overexpressing type 1 ASPL-TFE3 fusion gene, wild-type TFE3 and wild-type MCA205 tumors to PD-L1 treatment. Figure 4 As shown in Figure A, MCA205 tumors overexpressing the type 1 ASPL-TFE3 fusion gene are responsive to PD-L1. Figure 4 Middle B is the growth curve of the response of overexpression type 2 ASPL-TFE3 fusion gene, wild-type TFE3 and wild-type MCA205 tumors to PD-L1 treatment. Figure 4 As shown in Figure B, MCA205 tumors overexpressing the type 2 ASPL-TFE3 fusion gene are responsive to PD-L1. Figure 4 Middle C is the growth curve of the response of tumors overexpressing type 1 ASPL-TFE3 fusion gene, wild-type TFE3 and wild-type TC-1 to PD-L1 treatment. Figure 4 As shown in Figure C, TC-1 tumors overexpressing the type 1 ASPL-TFE3 fusion gene are responsive to PD-L1. Figure 4 D in the middle is the growth curve of the response of tumors overexpressing type 2 ASPL-TFE3 fusion gene, wild-type TFE3 and wild-type TC-1 to PD-L1 treatment. Figure 4 As shown in Figure D, TC-1 tumors that overexpress type 2 ASPL-TFE3 fusion gene respond to PD-L1; Figure 4 The results showed that tumors overexpressing the ASPL-TFE3 fusion gene responded better to PD-L1 treatment than the control group.

[0119] Figure 5It shows that ASPL-TFE3 fusion gene overexpressing tumors have CD8 + T cell infiltration; Figure 5 A in the middle shows the CD8 expression in MCA205 and TC-1 tumors overexpressing ASPL-TFE3 fusion gene after PD-L1 treatment. + T cell immunohistochemical staining results, from Figure 5 As shown in Figure A, PD-L1 treatment can increase CD8 + T cell infiltration; Figure 5 Middle B shows the MCA205 tumor overexpressing ASPL-TFE3 fusion gene after PD-L1 treatment, CD8 + T cell staining score statistics, from Figure 5 As shown in Figure B, PD-L1 treatment can increase the CD8 + T cell infiltration; Figure 5 Middle C is the CD8 + T cell staining score statistics, from Figure 5 As shown in Figure C, PD-L1 treatment can increase CD8 + T cell infiltration. Figure 5 The results showed that tumors overexpressing the ASPL-TFE3 fusion gene had a negative effect on CD8 + The T cell infiltration ratio was higher than that in the control group.

[0120] Figure 5 In panels B and C, “1” represents “WT”, “2” represents “TFE3”, “3” represents “fusion protein 1”, “4” represents “fusion protein 2”, “5” represents “WT and PD-L1”, “6” represents “TFE3 and PD-L1”, “7” represents “fusion protein 1 and PD-L1”, and “8” represents “fusion protein 2 and PD-L1”.

[0121] In summary, the present invention designs a new immunotherapy regimen based on the changes in tumor autophagy levels mediated by the ASPL-TFE3 fusion gene and the tumor microenvironment regulation mechanism, which is expected to solve the clinical problems of high drug resistance and limited efficacy in current tumor treatment.

[0122] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. An ASPL-TFE3 fusion gene, characterized in that The nucleotide sequence of the fusion gene is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. An expression vector, characterized in that: The expression vector contains the nucleotide sequence of the ASPL-TFE3 fusion gene according to claim 1.

3. A recombinant virus, characterized in that The recombinant virus carries the nucleotide sequence of the ASPL-TFE3 fusion gene according to claim 1; and the recombinant virus is a lentivirus.

4. Use of the ASPL-TFE3 fusion gene according to claim 1 in the preparation of a drug for enhancing the efficacy of immune checkpoint inhibitors, characterized in that: The immune checkpoint inhibitor is a PD-L1 antibody.

5. The use according to claim 4, characterized in that The ASPL-TFE3 fusion gene is introduced into tumor cells through a viral vector to express the ASPL-TFE3 fusion protein.

6. The use according to claim 5, characterized in that After the tumor cells express the ASPL-TFE3 fusion protein, immune activation and anti-tumor effects are promoted.

7. The use according to claim 5, characterized in that The ASPL-TFE3 fusion protein enhances the autophagy level of tumor cells.

8. The use according to claim 5, characterized in that The ASPL-TFE3 fusion protein increases CD8 + T cell infiltration ratio.

9. The use according to claim 4, characterized in that The tumor is a fibrosarcoma or a lung epithelial tumor.

10. A composition for treating tumors, characterized in that: The composition comprises the ASPL-TFE3 fusion gene according to claim 1, the expression vector according to claim 2 or the recombinant virus according to claim 3.

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