Application of ASPL-TFE3 fusion gene in the preparation of drugs that enhance the efficacy of immune checkpoint inhibitors

Abstract

This invention provides the application of the ASPL-TFE3 fusion gene in the preparation of drugs that enhance the efficacy of immune checkpoint inhibitors. The nucleotide sequence of the fusion gene is shown in SEQ ID NO:1 or SEQ ID NO:2. This invention relates to the field of tumor immunotherapy. This invention utilizes the ASPL-TFE3 fusion protein to regulate TFE3 expression and activity to enhance tumor cell autophagy and immune cell penetration, thereby improving the efficacy of immune checkpoint inhibitors in treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy, specifically involving the application of the ASPL-TFE3 fusion gene in the preparation of drugs that enhance the efficacy of immune checkpoint inhibitors. Background Technology

[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 mainly occurs in adolescents and young adults, with a 5-year survival rate of only 20%-46%. It is prone to lung and brain metastases, has poor response to traditional chemotherapy, and a high recurrence rate after surgical resection. Its molecular characteristic is the chromosomal unbalanced translocation der(17)t(X;17)(p11.2;q25), leading to the formation of the ASPL-TFE3 fusion gene.

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

[0004] For a long time, tumor cells have been the primary research focus in the fight against malignant tumors. Tumorigenesis is a progressive pathological process involving multiple genes, factors, and stages, and is related to abnormal cell proliferation, differentiation, apoptosis, and the activation of numerous proto-oncogenes and the abnormal expression of tumor suppressor genes. Malignant tumors impose a huge economic and social burden on both developed and underdeveloped countries, making the search for effective targeted therapies crucial for cancer treatment.

[0005] Therefore, designing a novel immunotherapy strategy based on the alteration of tumor autophagy levels and the regulation mechanism of the tumor microenvironment mediated by the ASPL-TFE3 fusion gene has important application prospects in current cancer treatment. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide the application of the ASPL-TFE3 fusion gene in the preparation of drugs that enhance the efficacy of immune checkpoint inhibitors. This invention utilizes a novel strategy to enhance tumor cell autophagy and activate the immune response, thereby improving the therapeutic efficacy of PD-L1 antibody therapy and enhancing the clinical treatment outcomes of tumors such as alveolar soft tissue sarcoma.

[0007] To achieve this objective, the present invention adopts the following technical solution:

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

[0009] This invention relates to the field of tumor immunotherapy. The invention describes a method that uses the ASPL-TFE3 fusion protein to regulate the expression and activity of TFE3 to enhance tumor cell autophagy and immune cell penetration, thereby improving the efficacy of immune checkpoint inhibitor therapy for tumors.

[0010] Based on the alteration of tumor autophagy levels mediated by the ASPL-TFE3 fusion gene and the regulation mechanism of the tumor microenvironment, this invention designs a novel immunotherapy regimen that is expected to solve the clinical challenges of high drug resistance and limited efficacy in current tumor treatment.

[0011] In one 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 finds that it can significantly enhance the autophagy level of tumor cells and tumor tissues.

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

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

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

[0015] Fourthly, the present invention provides the application of the ASPL-TFE3 fusion gene described in the first aspect in the preparation of a drug that enhances 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, the tumor cells express the ASPL-TFE3 fusion protein, which promotes immune activation and anti-tumor effects.

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

[0019] Preferably, the PD-L1 treatment increases CD8+ expression in tumors with increased ASPL-TFE3 fusion protein expression. + T-cell infiltration.

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

[0021] In one specific embodiment of the present invention, an ASPL-TFE3 fusion protein expression tumor model was constructed, which responded to PD-L1 treatment. PD-L1 treatment increased CD8+ expression in tumors containing ASPL-TFE3 fusion protein. + T cell infiltration; compared with wild-type TFE3 overexpression or wild-type tumor cell control groups, the ASPL-TFE3 fusion protein promoted immune activation and anti-tumor effects. Based on this, the present invention provides a novel strategy to enhance tumor cell autophagy and activate immune responses by regulating TFE3 protein expression through the ASPL-TFE3 fusion gene, thereby improving the efficacy of PD-L1 antibody therapy. This strategy has significant application prospects in tumor treatment.

[0022] Fifthly, the present invention provides a composition for treating tumors, the composition 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 includes an immune checkpoint inhibitor.

[0024] Preferably, the composition further includes 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 the ASPL-TFE3 fusion gene in MCA205 and TC-1 cells significantly enhances autophagy. PD-L1 treatment of tumor cells expressing the ASPL-TFE3 fusion protein increases CD8+ levels in the tumor. + T cell infiltration; compared with wild-type TFE3 overexpression or wild-type tumor cell control group, ASPL-TFE3 fusion protein has the effects of promoting immune activation and anti-tumor activity.

[0027] (2) Based on the alteration of tumor autophagy level mediated by the ASPL-TFE3 fusion gene and the tumor microenvironment regulation mechanism, this invention designs a novel immunotherapy regimen, which is expected to solve the current clinical problems of high drug resistance and limited efficacy in tumor treatment. Attached Figure Description

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

[0029] Figure 2 This is the result of immunohistochemical staining.

[0030] Figure 3 The image shows the results of immunohistochemical staining of LC3 to observe the level of autophagy in tumor tissue.

[0031] Figure 4 This study examines the response of ASPL-TFE3 fusion gene overexpressing tumors to PD-L1 antibody therapy.

[0032] Figure 5 CD8+ in ASPL-TFE3 fusion gene overexpressing tumors after PD-L1 antibody therapy + T cell infiltration status. Detailed Implementation

[0033] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0035] Example 1

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

[0037] To explore the effects of the ASPL-TFE3 fusion protein on autophagy and tumor growth, this embodiment constructed an ASPL-TFE3 fusion protein overexpression cell line and conducted mouse experiments to observe the effect of the ASPL-TFE3 fusion protein on tumor growth.

[0038] 1. Using lentiviral technology, construct cell lines overexpressing the ASPL-TFE3 fusion gene and wild-type TFE3.

[0039] (1) Find the protein coding region (CDS) to be overexpressed in NCBI, and then add the appropriate restriction site and have it synthesized by Suzhou Genewiz Company.

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

[0041]

[0042] The nucleotide sequence corresponding to the 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 approximately 60%-70%. The target plasmid and packaging plasmids psPAX2 and pMD2.G were prepared at a ratio of 4:3:1 to a concentration of 2 μg / well. This mixture was then diluted with 200 μL of OPTI, followed by the addition of 8 μL of PEI. The mixture was thoroughly mixed and allowed to stand for 15 minutes. After standing, the mixture was added to one well of a 6-well plate. The medium in the 6-well plate was changed after 4-6 hours. Lentiviral virus was collected after 48 hours, filtered through a 0.45 μM filter, aliquoted, and stored at -80°C, or immediately concentrated before infecting target cells.

[0054] (3) Infect the target cells with the packaged lentivirus. After 48 hours of infection, enrich the fluorescently labeled cells with a flow cytometer 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 to full capacity, the protein expression was verified at the protein level by Western blotting.

[0056] 2. Immunoblotting assay to detect autophagy levels

[0057] (1) Preparation of protein samples

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

[0059] Protein extraction: Resuspend the cell pellet in lysis buffer on ice for 30 minutes, centrifuge at 12,000 rpm, 4°C for 10 minutes. After centrifugation, collect a few volumes of supernatant and place them in a new EP tube. Add 4× SDS loading buffer, mix thoroughly, and heat in a 100°C water bath for 10 minutes. Store the collected protein samples at -20°C or use immediately.

[0060] (2) Preparation of polyacrylamide gel

[0061] Prepare the gel according to the instructions for use with Yake polyacrylamide gel.

[0062] (3) Sample loading and gel running

[0063] Add the samples to the wells in the designed sample order, using 26616 protein molecular weight standards (markers) on both sides. After sample loading, run the gel at 80 V. Once the samples have passed through the upper stacking gel, adjust the voltage to 120 V.

[0064] (4) Transfer membrane

[0065] Activate the PVDF membrane with methanol. Remove the protein gel block and cut off the stacking gel. Then, place the following items in the transfer clamp in the following order: sponge-filter paper-gel-PVDF membrane-filter paper-sponge, removing any air bubbles. Finally, place the transfer clamp in the transfer tank, add two ice cubes, pour in the transfer buffer, and close the lid. Set the current to 320 mA and the time to 2 hours to begin the transfer.

[0066] (5) Skim milk sealing

[0067] After the transfer was completed, 5% skim milk was prepared using TBST as the blocking solution. The PVDF membrane was placed in the blocking solution and sealed at room temperature for 1 hour.

[0068] (6) Primary antibody incubation

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

[0070] (7) Washing the film: TBST quick wash 3 times, 15 minutes each time.

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

[0072] (9) Washing the film: TBST quick wash 3 times, 15 minutes each time.

[0073] (10) Development: The PVDF film is wetted with the luminescent mixture and then exposed using a fluorescence chemiluminescence imaging system.

[0074] 3. Mouse subcutaneous xenograft model

[0075] (1) Mouse feeding and grouping

[0076] All animals were housed in designated pathogen-free facilities. All animal studies were reviewed and approved by the Animal Care and Use Institutional Committee of the Suzhou Institute of Systems Medicine. C57BL / 6N (female, 6-8 weeks old, weighing 18-20 g) were purchased from Zhejiang Vital River Co., Ltd. Mice were randomly assigned to groups of 5 mice each prior to inoculation.

[0077] (2) Cell treatment

[0078] Tumor cells (MCA205-WT, MCA205-TFE3, MCA205-ASPL-TFE3, TC-1-WT, TC-1-TFE3, TC-1-ASPL-TFE3) were digested to prepare single-cell suspensions. The cells were washed twice with PBS, counted, and then the cell concentration was adjusted to 2 × 10⁻⁶ cells / cells. 6 Cells were resuspended in PBS solution at 100 μL for tumor loading and later use.

[0079] (3) Tumor growth detection

[0080] Tumor cells were inoculated into the right back of C57BL / 6N mice, with each mouse receiving 2 × 10⁶ cells. 6 Each cell. Starting from day 7, the tumor size was measured every 2-3 days using calipers, and the tumor area was calculated by multiplying the length by the width. When the tumor area exceeded 300 mm, the tumor was considered a tumor. 2This refers to euthanizing mice.

[0081] 4. Immunohistochemical experiments on tumor tissue

[0082] (1) Preparation of frozen section samples

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

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

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

[0086] (2) Fluorescent staining

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

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

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

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

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

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

[0093] After the secondary antibody incubation is complete, wash the slides three times with PBS, letting them stand for about 5 minutes each time.

[0094] Staining cell nuclei: Dilute Hochest (Herster, Thermo Fisher Scientific, H3569) dye (1:1000) with PBS and stain the sections for 15 minutes.

[0095] After washing three times with PBS, letting it stand for about 5 minutes each time, then adding an anti-quenching agent and mounting the slide, it was then taken to a platform microscope for imaging.

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

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

[0098] (1) Mouse model construction

[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 backs of C57BL / 6N mice, with each mouse receiving 2 × 10⁻⁶ cells. 6 Seven days after tumor implantation, mice were injected with PD-L1 antibody via the tail vein at a dose of 200 μg per mouse, every other day for a total of three injections. The control group was treated with PBS. Starting from day 7, tumor size was measured using calipers every 2-3 days.

[0100] The above experiments show that PD-L1 antibody can effectively control the growth of tumors expressing ASPL-TFE3 fusion protein.

[0101] (2) Immunohistochemical CD8 staining experiment of tumor tissue

[0102] Preparation of frozen section samples: The tumor tissue was surgically separated, 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) for dehydration for 24-48 hours, and observed until the tumor tissue completely sank to the bottom. The tumor tissue was then retrieved, dried, and embedded using OCT embedding agent, and then frozen at -20°C.

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

[0105] Fluorescent staining: After the frozen tissue sections have been brought to room temperature, they are washed with PBS to remove the embedding agent.

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

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

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

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

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

[0111] After the secondary antibody incubation is complete, wash the slides three times with PBS, letting them stand for about 5 minutes each time.

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

[0113] After washing three times with PBS, letting it stand for about 5 minutes each time, then adding an anti-quenching agent and mounting the slide, it was then taken to a platform microscope for imaging.

[0114] Figure 1 This study demonstrates the effect of overexpression of the ASPL-TFE3 fusion gene in wild-type MCA205 and TC-1 tumor cells using lentiviral technology on autophagy levels and tumor growth. Figure 1 Figures A and D illustrate that the ASPL-TFE3 fusion gene can enhance the autophagy level of tumor cells; Figure 1 The BC and EF diagrams in the middle illustrate that the ASPL-TFE3 fusion gene can promote tumor growth.

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

[0116] Figure 2 The results of immunohistochemical staining show that the ASPL-TFE3 fusion gene can increase the autophagy level in tumor tissue.

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

[0118] Figure 4 The study showed the response of ASPL-TFE3 fusion gene overexpressing tumors to PD-L1 antibody therapy; Figure 4 In section A, growth curves of tumors overexpressing the type 1 ASPL-TFE3 fusion gene, wild-type TFE3, and wild-type MCA205 in response to PD-L1 therapy are shown. Figure 4 As shown in Figure A, tumors that overexpress the type 1 ASPL-TFE3 fusion gene MCA205 respond to PD-L1. Figure 4 In section B, growth curves of tumors overexpressing the type2 ASPL-TFE3 fusion gene, wild-type TFE3, and wild-type MCA205 in response to PD-L1 therapy are shown. Figure 4 As shown in B, tumors that overexpress the type2 ASPL-TFE3 fusion gene MCA205 respond to PD-L1. Figure 4 In the middle, C represents the growth curves of tumors overexpressing the type 1 ASPL-TFE3 fusion gene, wild-type TFE3, and wild-type TC-1 in response to PD-L1 therapy. Figure 4 As can be seen from C, tumors that overexpress the type1 ASPL-TFE3 fusion gene TC-1 respond to PD-L1; Figure 4 The curves in D represent the growth curves of tumors overexpressing the type2 ASPL-TFE3 fusion gene, wild-type TFE3, and wild-type TC-1 in response to PD-L1 therapy. Figure 4 As can be seen from D, tumors overexpressing the type2 ASPL-TFE3 fusion gene TC-1 respond to PD-L1; from 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 5This demonstrates the CD8 activity in tumors overexpressing the ASPL-TFE3 fusion gene after PD-L1 antibody therapy. + T cell infiltration status; Figure 5 In the middle A group, tumors overexpressing the ASPL-TFE3 fusion gene MCA205 and TC-1 showed increased CD8 levels after PD-L1 treatment. + Immunohistochemical results of T cell staining, from Figure 5 As shown in Figure A, PD-L1 therapy can increase CD8 expression in tumors that overexpress the ASPL-TFE3 fusion gene MCA205 and TC-1. + T cell infiltration; Figure 5 In tumor B, which overexpresses the ASPL-TFE3 fusion gene MCA205, CD8 levels were observed after PD-L1 therapy. + A statistical chart of T cell staining scores, from Figure 5 As shown in the B-cell assay, PD-L1 therapy can increase CD8+ expression in tumors that overexpress the ASPL-TFE3 fusion gene MCA205. + T cell infiltration; Figure 5 In the middle C, TC-1 tumors overexpressing the ASPL-TFE3 fusion gene were observed after PD-L1 treatment, with CD8... + A statistical chart of T cell staining scores, from Figure 5 As can be seen from the data in the middle section, PD-L1 therapy can increase CD8 in tumors that overexpress the ASPL-TFE3 fusion gene TC-1. + T cell infiltration. From Figure 5 The results showed that tumors overexpressing the ASPL-TFE3 fusion gene had increased CD8 counts after PD-L1 treatment. + The proportion of T-cell infiltration was higher than that in the control group.

[0120] Figure 5 In 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, this invention designs a novel immunotherapy regimen based on the alteration of tumor autophagy levels mediated by the ASPL-TFE3 fusion gene and the regulation mechanism of the tumor microenvironment, which is expected to solve the clinical challenges of high drug resistance and limited efficacy in current tumor treatment.

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

Claims

1. The application of the ASPL-TFE3 fusion gene in the preparation of drugs that enhance the efficacy of immune checkpoint inhibitors, characterized in that, The immune checkpoint inhibitor is a PD-L1 antibody; the drug is used to treat fibrosarcoma or lung epithelial tumors. The nucleotide sequence of the fusion gene is shown in SEQ ID NO:1 or SEQ ID NO:2; The ASPL-TFE3 fusion gene was introduced into tumor cells via a viral vector to express the ASPL-TFE3 fusion protein.

2. The application according to claim 1, characterized in that, After the tumor cells expressed the ASPL-TFE3 fusion protein, they promoted immune activation and anti-tumor effects.

3. The application according to claim 1, characterized in that, The ASPL-TFE3 fusion protein enhances the autophagy level of tumor cells.

4. The application according to claim 1, characterized in that, The ASPL-TFE3 fusion protein increases CD8 + T cell infiltration rate.

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