Use of an oncolytic adenovirus with ifn alpha 2b in the preparation of a medicament for treating melanoma

Abstract

The application discloses an oncolytic adenovirus and IFN alpha 2b in the preparation of a drug for treating melanoma, and relates to the technical field of biological medicine.The application first proposes that the oncolytic adenovirus combined with IFN alpha 2b has a significant synergistic tumor inhibition effect on treating melanoma, which is specifically embodied in that in a cell experiment, the oncolytic adenovirus combined with IFN alpha 2b can inhibit the growth of melanoma cells and promote the apoptosis of the melanoma cells; in an animal experiment, the oncolytic adenovirus combined with IFN alpha 2b can significantly inhibit the growth of in situ melanoma, promote the apoptosis of tumor cells, prolong the survival period and have good biocompatibility.The combined application of the oncolytic adenovirus and IFN alpha 2b has a potential application prospect for treating melanoma, and can provide a more effective treatment scheme for melanoma patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of an oncolytic adenovirus and IFNα2b in the preparation of a drug for treating melanoma. Background Technology

[0002] Malignant melanoma is the fifth most common cancer in humans. Originating from melanocytes, it develops rapidly, metastasizes early and widely, has an extremely poor prognosis, and a high mortality rate. Different treatment approaches are adopted depending on the stage of melanoma. Clinically, surgical resection is the first-line treatment for melanoma. However, for patients with intermediate or advanced melanoma, surgical treatment cannot completely eradicate the tumor and carries a high risk of recurrence. Therefore, adjuvant therapy is necessary to combat tumor development, progression, and recurrence / metastasis. Currently, the main adjuvant therapies for melanoma include chemotherapy, radiotherapy, targeted therapy, and immunotherapy. In recent years, molecularly targeted drugs and immune checkpoint inhibitors have been successfully applied to adjuvant therapy after melanoma surgery and systemic treatment for advanced stages.

[0003] Interferon (IFN) is a cytokine with antiviral, antitumor, and immunomodulatory effects. Currently, IFNα2b has been used to treat various tumors, including leukemia, myeloma, renal cell carcinoma, and melanoma. Clinical data shows that IFNα2b can reduce postoperative recurrence and control tumor progression in cancer patients. High-dose IFNα-2b has been approved in the United States for adjuvant therapy in stage IIB and III melanoma; however, adverse reactions such as neutropenia, liver toxicity, and fatigue are not negligible. Furthermore, studies have reported that continuous administration of interferon may induce CD8+ T cell depletion, impairing antitumor immunotherapy and resulting in poor efficacy of IFNα2b monotherapy in clinical practice. Therefore, we need to explore a new immunotherapy approach based on IFNα2b, which has important guiding significance for improving the treatment efficacy of melanoma.

[0004] Oncolytic viruses are natural or genetically modified viruses characterized by their selective ability to infect and kill tumor cells while causing minimal damage to normal cells. These viruses exert their anti-tumor effects primarily by directly lysing tumor cells and indirectly enhancing the host's anti-tumor immunity. Studies have shown that their combination with chemotherapy, radiotherapy, and PD-1 inhibitors has a synergistic effect in treating tumors. Currently, there are no reported studies in this field on the combined use of oncolytic viruses and IFNα2b for the treatment of melanoma. Summary of the Invention

[0005] The purpose of this invention is to provide an application of oncolytic adenovirus and IFNα2b in the preparation of drugs for treating melanoma, thereby addressing the problems existing in the prior art. This invention has found that oncolytic adenovirus combined with IFNα2b has a significant synergistic tumor-suppressive effect in the treatment of melanoma, which has potential application prospects for melanoma treatment and can provide more effective treatment options for melanoma patients.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention provides a composition for treating melanoma, comprising oncolytic adenovirus and IFNα2b;

[0008] The oncolytic adenovirus has a human telomerase promoter and the E1A gene, which controls adenovirus replication initiation.

[0009] Furthermore, the ratio of the oncolytic adenovirus to the IFNα2b is 10:1. 8 PFU: 10 4 U.

[0010] Furthermore, the method for constructing the oncolytic adenovirus includes the following steps:

[0011] The human telomerase promoter hTERT was replaced with the promoter CMV in the shuttle plasmid pDC315 of the recombinant adenovirus dual plasmid system to construct the pDC315-hTERT-3E plasmid.

[0012] Using the adenovirus replication initiation control gene E1A cDNA fragment as a template, EcoRI and BamHI restriction sites were introduced into the primers, and the E1A gene fragment was amplified by PCR.

[0013] The EcoRI and BamHI double-digested fragments of the pDC315-hTERT-3E plasmid were ligated with the E1A gene fragment to construct the shuttle plasmid pDC315-hTERT-3E-E1A.

[0014] The shuttle plasmid pDC315-hTERT-3E-E1A and the backbone plasmid pBGHfrtΔE1,3FLP were co-transfected into HEK293 cells at a mass ratio of 1:2. After culture, the cells were purified to obtain the oncolytic adenovirus.

[0015] The present invention also provides the use of the above-described composition in the preparation of a medicament for treating melanoma.

[0016] Furthermore, the composition exerts a therapeutic effect on melanoma by inhibiting the growth of melanoma cells and promoting apoptosis of melanoma cells.

[0017] The present invention also provides the use of oncolytic adenovirus in the preparation of drugs for treating melanoma, wherein the oncolytic adenovirus has a human telomerase promoter and an adenovirus replication initiation control E1A gene.

[0018] Furthermore, the method for constructing the oncolytic adenovirus includes the following steps:

[0019] The human telomerase promoter hTERT was replaced with the promoter CMV in the shuttle plasmid pDC315 of the recombinant adenovirus dual plasmid system to construct the pDC315-hTERT-3E plasmid.

[0020] Using the adenovirus replication initiation control gene E1A cDNA fragment as a template, EcoRI and BamHI restriction sites were introduced into the primers, and the E1A gene fragment was amplified by PCR.

[0021] The EcoRI and BamHI double-digested fragments of the pDC315-hTERT-3E plasmid were ligated with the E1A gene fragment to construct the shuttle plasmid pDC315-hTERT-3E-E1A.

[0022] The shuttle plasmid pDC315-hTERT-3E-E1A and the backbone plasmid pBGHfrtΔE1,3FLP were co-transfected into HEK293 cells at a mass ratio of 1:2. After culture, the cells were purified to obtain the oncolytic adenovirus.

[0023] The present invention also provides a medicament for treating melanoma, wherein the active ingredient comprises oncolytic adenovirus or the above-described composition;

[0024] The oncolytic adenovirus has a human telomerase promoter and the E1A gene, which controls adenovirus replication initiation.

[0025] Furthermore, the method for constructing the oncolytic adenovirus includes the following steps:

[0026] The human telomerase promoter hTERT was replaced with the promoter CMV in the shuttle plasmid pDC315 of the recombinant adenovirus dual plasmid system to construct the pDC315-hTERT-3E plasmid.

[0027] Using the adenovirus replication initiation control gene E1A cDNA fragment as a template, EcoRI and BamHI restriction sites were introduced into the primers, and the E1A gene fragment was amplified by PCR.

[0028] The EcoRI and BamHI double-digested fragments of the pDC315-hTERT-3E plasmid were ligated with the E1A gene fragment to construct the shuttle plasmid pDC315-hTERT-3E-E1A.

[0029] The shuttle plasmid pDC315-hTERT-3E-E1A and the backbone plasmid pBGHfrtΔE1,3FLP were co-transfected into HEK293 cells at a mass ratio of 1:2. After culture, the cells were purified to obtain the oncolytic adenovirus.

[0030] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0031] The present invention discloses the following technical effects:

[0032] This invention is the first to propose that oncolytic adenovirus combined with IFNα2b has a significant synergistic tumor-suppressive effect in the treatment of melanoma. Specifically, in cell experiments, oncolytic adenovirus combined with IFNα2b can inhibit the growth of melanoma cells and promote their apoptosis; in animal experiments, oncolytic adenovirus combined with IFNα2b can significantly inhibit the growth of in situ melanoma, promote tumor cell apoptosis, prolong survival, and exhibit good biocompatibility. The combination of oncolytic virus and IFNα2b has potential application prospects in the treatment of melanoma and could provide more effective treatment options for melanoma patients. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram of the dosing regimen for B16-F10 tumor-bearing mice;

[0035] Figure 2 The images (A) and (B) show the Ki67 immunohistochemical staining of the tumor dissected after the experiment in Example 1.

[0036] Figure 3 The images shown are TUNEL immunohistochemical staining images (A) and quantitative analysis results (B) of the tumor dissected after the experiment in Example 1.

[0037] Figure 4 Flow cytometry atlases (A) and quantitative analysis results (B) of apoptosis in B16-F10 cells from different treatment groups in Example 2; n=3;

[0038] Figure 5 This is a tumor growth curve of B16-F10 tumor-bearing mice in different treatment groups in Example 3; n=5;

[0039] Figure 6These are photographs of tumors dissected after the experiments in different treatment groups in Example 3; n = 4;

[0040] Figure 7 Survival curves of B16-F10 tumor-bearing mice in different treatment groups in Example 3; n=5;

[0041] Figure 8 This is a graph showing the weight changes of B16-F10 tumor-bearing mice in different treatment groups in Example 3; n = 5;

[0042] Figure 9 The image shows H&E staining of the major organs (heart, liver, spleen, lung, and kidney) of tumor-bearing mice in different treatment groups (B10-F10) in Example 3. Detailed Implementation

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0046] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0047] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0048] C57BL / 6 mice (6-8 weeks old) were purchased from the Experimental Animal Center of Chongqing Medical University and housed in the SPF experimental animal facility. All animal experiments were conducted in accordance with international guidelines for animal experiments.

[0049] All quantitative experimental data were obtained at least three times independently and presented as mean ± standard error (Mean ± SEM). Significance analysis was performed using GraphPad Prism (8.0.1) software via unpaired two-tailed t-tests. Unless otherwise stated, P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) was considered statistically significant.

[0050] The oncolytic adenvirus (OA) referred to in this invention is a selectively replicating adenvirus possessing a human telomerase promoter and an E1A gene controlling adenvirus replication initiation. By inserting a telomerase promoter sequence before the E1A gene controlling adenvirus replication initiation, an oncolytic adenvirus with tumor cell-specific replication capability was constructed (disclosed in Chinese patent CN201811026737.5). The construction method is detailed below:

[0051] 1) Construction of pDC315-hTERT-3E-E1A adenovirus plasmid:

[0052] a. Replace the CMV promoter in the shuttle plasmid pDC315 of the recombinant adenovirus dual plasmid system with the human telomerase promoter (hTERT) to construct the pDC315-hTERT-3E plasmid.

[0053] b. Using the adenovirus replication initiation control gene E1A cDNA fragment as a template, EcoRI and BamHI restriction sites were introduced into the primers, and E1A was amplified by PCR.

[0054] c. The EcoRI and BamHI double digestion fragments of plasmid pDC315-hTERT-3E were ligated with the E1A gene fragment to construct the shuttle plasmid pDC315-hTERT-3E-E1A.

[0055] 2) The shuttle plasmid pDC315-hTERT-3E-E1A and the backbone plasmid pBGHfrtΔE1,3FLP were co-transfected into HEK293 cells at a mass ratio of 1:2.

[0056] 3) After transfection, change the culture medium every 2 days and continuously observe the cell status;

[0057] 4) One week after transfection, cell plaques can be seen. When the cells are almost detached from the culture dish, collect the cells and culture supernatant.

[0058] 5) Purify the oncolytic adenovirus according to the operating procedures provided in the Vivapure AdenoPACK 20RT adenovirus purification kit. Store the purified oncolytic adenovirus at -80°C for later use.

[0059] When OA infects normal cells, the virus cannot initiate replication properly and is quickly cleared by the cells due to the low content and activity of telomerase in normal cells, thus causing no toxicity to the cells. However, when OA infects tumor cells, the tumor cells contain highly active telomerase, allowing the virus to replicate rapidly and produce a large number of progeny viruses. These progeny viruses lyse the infected tumor cells, thereby killing them.

[0060] The culture method for oncolytic adenovirus is as follows: 1 mL of virus seed solution was inoculated into a 10 cm culture dish containing a monolayer of HEK293T cells and cultured at 37°C in a 5% CO2 incubator for 1 hour. The original solution was discarded, and 10 mL of fresh DMEM virus maintenance medium containing 2% FBS was added. The cells were then cultured at 37°C in a 5% CO2 incubator. Daily observations were performed. When all HEK293T cells showed cytopathic effects, the cells were subjected to three freeze-thaw cycles in liquid nitrogen, centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected and aliquoted for use. A final yield of 1 × 10⁶ cells was obtained. 9 Oncolytic adenovirus at a titer of VPs / mL.

[0061] The specific culture method for the mouse melanoma cell line B16-F10 is as follows: Cells were cultured in 10cm cell culture dishes containing 10% FBS, 1% penicillin-streptomycin (100U / mL penicillin, 100μg / mL streptomycin), and 1% glutamine in RPMI 1640 medium. The dishes were placed in a constant temperature incubator at 37℃, 5% CO2, and 95% relative humidity. When the cell confluence reached 80%, the medium was discarded, and the cells were washed twice with PBS. The cells were then digested for 1 min with trypsin containing 0.25% EDTA, and the digestion was terminated by adding an equal volume of complete medium. The cell suspension was collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in complete medium and passaged at a 1:5 ratio.

[0062] Example 1

[0063] 1. Establishment of a tumor-bearing mouse model

[0064] B16-F10 tumor cells with 80% confluence were digested with trypsin containing 0.25% EDTA, centrifuged and the supernatant was discarded. The cells were washed once with PBS, resuspended in serum-free RPMI-1640 medium, and the cell density was adjusted to 1×10⁶ cells / year. 5 A tumor-bearing mouse model was established by inoculating 100 μL of the sample onto the back of C57BL / 6 mice at a dose of 1 / mL.

[0065] 2. Experimental grouping and treatment

[0066] Tumor-bearing mice were randomly divided into four groups of five mice each. Figure 1 The dosing regimens shown were PBS, OA, IFNα2b, and IFNα2b+OA, respectively, with the OA dose being 1×10⁻⁶. 8 PFU / animal, IFNα2b dosage is 1×10 4 U / only.

[0067] (1) PBS group: when the tumor volume reached 100 mm 3 Afterwards, each mouse was subcutaneously injected with 100 μL of PBS, and the administration was repeated every other day for two consecutive times.

[0068] (2) OA group: when the tumor volume reaches 100 mm 3 Subsequently, each mouse was injected intratumorally with 1×10 8 PFU OA, administered every other day, twice consecutively;

[0069] (3) IFNα2b group: when the tumor volume reached 100 mm 3 Afterwards, each mouse was subcutaneously injected with 1×10 4 U's IFNα2b, administered every other day, twice consecutively;

[0070] (4) IFNα2b+OA group: when the tumor volume reached 100 mm 3 Subsequently, each mouse was injected intratumorally with 1×10 8 PFU OA and subcutaneous injection 1×10 4 U's IFNα2b was administered every other day for two consecutive times.

[0071] 3. Immunohistochemical staining of Ki67 and TUNEL in tumor tissues of mice in each treatment group

[0072] To further investigate the differences in tumor growth in tumor-bearing mice after different treatments, mice were sacrificed by cervical dislocation after two consecutive administrations. The tumors were dissected, fixed in 4% paraformaldehyde for 24 hours, and prepared into 5μm paraffin sections. Ki67 and TUNEL immunohistochemical staining were performed as follows:

[0073] (1) The sections were sequentially treated with xylene I and II for 10 min each, anhydrous ethanol for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then washed with distilled water for 5 min to dewax to water.

[0074] (2) Immerse the sections in Tris-EDTA antigen retrieval solution (pH=9.0), microwave on high for 3 min and on low for 15 min for antigen retrieval, wash with PBS 3 times, 5 min each time;

[0075] (3) Immerse the slices in a solution containing 3% H2O2 and incubate at room temperature in the dark for 30 min to inactivate endogenous peroxidase. Wash with PBS 3 times, 5 min each time.

[0076] (4) Draw a circle around the tissue with an immunohistochemical pen, add diluted primary antibody, incubate overnight at 4°C, and wash 3 times with PBS for 5 minutes each time.

[0077] (5) Add the diluted HRP-labeled secondary antibody, incubate at room temperature in the dark for 1 h, and wash with PBS 3 times for 5 min each time;

[0078] (6) Add DAB color development solution and rinse the slide with tap water in time to stop the color development.

[0079] (7) Stain with hematoxylin for 5 min, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for 3 s, rinse with tap water, hematoxylin blue for 10 s, rinse with tap water.

[0080] (8) The slices were sequentially treated with 70% ethanol for 5 min, 80% ethanol for 5 min, 90% ethanol for 5 min, 95% ethanol for 5 min, anhydrous ethanol for 10 min, and xylene I and II for 10 min each to dehydrate and clear the slices.

[0081] (9) After the sections are dried, they are mounted with neutral resin; images are acquired using a digital pathology slide scanner to analyze tumor cell proliferation and apoptosis.

[0082] The results are as follows Figure 2 and Figure 3 As shown in the figure, compared with the PBS group, the IFNα2b+OA group showed extensive apoptosis in tumor tissue, increased intercellular spaces, pyknosis of nuclei, and strong eosinophilic cytoplasm. Immunohistochemical results collectively indicate that the combined use of IFNα2b and OA can effectively induce apoptosis and necrosis in tumor tissue, thereby significantly inhibiting tumor growth.

[0083] Example 2

[0084] B16-F10 cells with 80% confluence were digested with trypsin containing 0.25% EDTA, and the supernatant was discarded by centrifugation. Tumor cells were diluted with complete culture medium and seeded into 6-well cell culture plates at 2 × 10⁶ cells per well. 5 Cells. Cell culture plates were incubated overnight at 37°C in a 5% CO2 incubator. The culture medium was discarded, and the cells were washed twice with PBS. The cells in the wells were then divided into four groups of n=3:

[0085] (1) PBS group, treated with PBS for 48 h;

[0086] (2) OA group, provide OA processing for 48 hours;

[0087] (3) IFNα2b group, treated with IFNα2b for 48 hours;

[0088] (4) IFNα2b+OA group, and OA and IFNα2b were administered simultaneously for 48h.

[0089] In (2) and (4), the dosage of OA was the same, which was 20 MOI; in (3) and (4), the dosage of IFNα2b was the same, which was 200 ng / mL.

[0090] The apoptosis levels of B16-F10 tumor cells after different treatments were detected by flow cytometry using the Annexin V-FITC / PI apoptosis detection kit. Specifically, after treatment with different drugs, the culture medium was discarded, and the cells were washed twice with PBS, digested with trypsin containing 0.25% EDTA, and collected. Each sample was resuspended in 100 μL of 1× Binding Buffer, and 5 μL of Annexin V-FITC and 5 μL of PI staining solution were added. The mixture was gently mixed and incubated at room temperature in the dark for 15 min. Then, 400 μL of 1× Binding Buffer was added, mixed, and the cells were passed through a 300-mesh sieve. The fluorescence intensity of FITC and PI in the cells was detected by flow cytometry. 10,000 cells were collected for each sample.

[0091] Depend on Figure 4 It can be seen that over 90% of B16-F10 cells in the PBS, OA, and IFNα2b groups were Annexin V / PI double-negative, indicating normal cells; while in the IFNα2b+OA group, 11% of B16-F10 cells were Annexin V / I double-positive, indicating late-stage apoptosis, and another 14% were Annexin V single-positive, indicating early-stage apoptosis. These results demonstrate that the combined use of IFNα2b and OA can effectively promote apoptosis in melanoma cells.

[0092] Example 3

[0093] Efficacy evaluation of drugs for B16-F10 melanoma

[0094] 1. Pharmacodynamic evaluation of tumor growth

[0095] A B16-F10 tumor-bearing mouse model was established according to the method in Example 1. C57BL / 6 mice were randomly divided into 4 groups of 5 mice each. Figure 1The dosing regimens included PBS, OA, IFNα2b, and IFNα2b+OA, with OA administered at a dose of 1×10⁻⁶. 8 PFU / animal, IFNα2b dosage is 1×10 4 U / mouse. Starting from the first administration, the major axis (L) and minor axis (W) of the mouse tumor were measured every other day using electronic vernier calipers. The tumor volume (V) was calculated according to the following formula, and a tumor growth curve was plotted:

[0096] V = 0.5 × L × W 2 .

[0097] The results are as follows Figure 5 As shown in the figure. Compared with the PBS group, both OA and IFNα2b administration alone have certain tumor-suppressing effects. This is because they each have certain regulatory effects on the tumor microenvironment and tumor tissue. The IFNα2b+OA group can significantly inhibit tumor growth, achieving a "1+1>2" effect.

[0098] On day 12 after drug administration, mice in each group were sacrificed by cervical dislocation, and tumors and major organs (heart, liver, spleen, lung, and kidney) were removed. The tumor tissue was photographed, and the results are as follows: Figure 6 As shown in the figure, compared with other groups, the tumor volume in the IFNα2b+OA group was significantly reduced, consistent with the tumor growth curve results, demonstrating a good effect of combined immunotherapy.

[0099] 2. Mouse survival analysis

[0100] The B16-F10 melanoma model was grouped and administered the same drugs as in section 1. The major and minor diameters of the mouse tumors were measured using electronic calipers, and the tumor volume was calculated. Once the tumor volume exceeded 1500 mm... 3 This means that the mouse is considered dead.

[0101] Survival results of different groups of B16-F10 tumor-bearing mice are as follows: Figure 7 As shown, all mice in the PBS group died on day 24 after tumor implantation; mice in the OA and IFNα2b groups began to die on day 9 after tumor implantation; and on day 27 after tumor implantation, 80% of the mice in the IFNα2b+OA group were still alive, indicating that the combination of IFNα2b and OA can significantly prolong the survival of B16-F10 tumor-bearing mice.

[0102] 3. In vivo safety analysis

[0103] Starting with the first dose in Part 1, the body weight of mice in each group was measured using an electronic balance the following day, and a curve of body weight change was plotted.

[0104] Changes in body weight of tumor-bearing mice during the experiment are as follows Figure 8As shown, no significant decrease in body weight was observed in any of the mouse groups, indicating that the combined administration of IFNα2b+OA has good biocompatibility.

[0105] Major organs (heart, liver, spleen, lung, and kidney) of tumor-bearing mice in different treatment groups were fixed in 4% paraformaldehyde for 24 hours to prepare 5 μm paraffin sections, and H&E staining was performed on the paraffin sections as follows:

[0106] (1) The sections were sequentially treated with xylene I and II for 10 min each, anhydrous ethanol for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then washed with distilled water for 5 min to dewax to water.

[0107] (2) Stain the sections with hematoxylin for 10 min and rinse with distilled water;

[0108] (3) Stain the sections in eosin solution for 3 minutes, then rinse with distilled water;

[0109] (4) The slices were sequentially treated with 70% ethanol for 5 min, 80% ethanol for 5 min, 90% ethanol for 5 min, 95% ethanol for 5 min, anhydrous ethanol for 10 min, and xylene I and II for 10 min each to dehydrate and clear the slices.

[0110] (5) After the sections are dried, they are mounted with neutral resin; images are acquired using a digital pathology slide scanner to observe the fine morphological structure of the tissue.

[0111] The H&E staining results of the major organs of mice in each group are as follows: Figure 9 As shown, after treatment with the drug, no obvious inflammation or microstructural damage was observed in the heart, liver, spleen, lungs, and kidneys of tumor-bearing mice, further demonstrating that IFNα2b+OA has good biocompatibility.

[0112] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A composition for treating melanoma, characterized in that, Including oncolytic adenovirus and IFNα2b; The oncolytic adenovirus has a human telomerase promoter and adenovirus replication initiation controls. E1A Gene; The ratio of the oncolytic adenovirus to the IFNα2b is 10:

1. 8 PFU: 10 4 U; The method for constructing the oncolytic adenovirus includes the following steps: The human telomerase promoter hTERT was replaced with the promoter CMV in the shuttle plasmid pDC315 of the recombinant adenovirus dual plasmid system to construct the pDC315-hTERT-3E plasmid. Adenovirus replication initiation control gene E1A Using a cDNA fragment as a template, EcoRI and BamHI restriction sites were introduced into the primers for PCR amplification. E1A Gene fragments; The EcoRI and BamHI double digestion fragment of the pDC315-hTERT-3E plasmid was combined with the... E1A Gene fragments were ligated to construct the shuttle plasmid pDC315-hTERT-3E-E1A; The shuttle plasmid pDC315-hTERT-3E-E1A and the backbone plasmid pBGHfrtΔE1,3FLP were co-transfected into HEK293 cells at a mass ratio of 1:

2. After culture, the cells were purified to obtain the oncolytic adenovirus.

2. Use of the composition as described in claim 1 in the preparation of a medicament for treating melanoma.

3. The application according to claim 2, characterized in that, The composition exerts its therapeutic effect on melanoma by inhibiting the growth of melanoma cells and promoting their apoptosis.

4. A drug for treating melanoma, characterized in that, The active ingredient includes the composition of claim 1.

5. The drug according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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