Application of oncolytic adenovirus and IFN alpha2b in preparation of medicine for treating melanoma

Abstract

The invention discloses an application of oncolytic adenovirus and IFN (interferon) alpha2b in preparation of a medicament for treating melanoma, and relates to the technical field of biological medicines. The invention firstly proposes that the oncolytic adenovirus combined IFN alpha2b has a remarkable synergistic tumor inhibition effect on treating melanoma, which is specifically reflected in that in a cell experiment, the oncolytic adenovirus combined IFN alpha2b can inhibit the growth of melanoma cells and promote the apoptosis of the melanoma cells; in animal experiments, the oncolytic adenovirus combined with IFN alpha2b can significantly inhibit the growth of in-situ melanoma, promote the apoptosis of tumor cells and prolong the lifetime, and has good biocompatibility. The oncolytic adenovirus and the IFN alpha2b are combined for application, the potential application prospect is achieved for treating the melanoma, and a more effective treatment scheme can be provided for patients with the melanoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an application of an oncolytic adenovirus and IFNα2b in preparing a drug for treating melanoma. Background Art

[0002] Malignant melanoma is the fifth most common tumor in humans. It originates from melanocytes, develops rapidly, metastasizes early and widely, has a very poor prognosis, and has a high mortality rate. Different treatment methods are adopted according to the different stages of melanoma. In clinical practice, surgical resection is the first choice for melanoma. However, for patients with advanced melanoma, surgical treatment cannot achieve tumor eradication and has a high risk of recurrence. Therefore, adjuvant therapy is needed to resist the occurrence, development, recurrence and metastasis of the tumor. At present, the main adjuvant treatments for melanoma include chemotherapy, radiotherapy, targeted therapy and immunotherapy. In recent years, anti-tumor drugs such as molecular targeted drugs and immune checkpoint inhibitors have been successfully used in postoperative adjuvant therapy and advanced systemic treatment of melanoma. However, the target gene mutation rate of targeted drug therapy for melanoma patients in my country is low, and the response rate of acral and mucosal melanoma to immune checkpoint inhibitors is low. The results of clinical studies completed in China show that the objective efficacy of second-line treatment of acral and mucosal melanoma with immune checkpoint inhibitors is only 14% to 18.5% and 0 to 13.3%.

[0003] Interferon (IFN) is a cytokine with antiviral, antitumor and immunomodulatory effects. Currently, IFNα2b has been used to treat a variety of tumors, including leukemia, myeloma, renal cell carcinoma, and melanoma. Clinical data show that IFNα2b can reduce postoperative recurrence and control tumor progression in tumor patients. High-dose IFNα-2b has been approved in the United States for adjuvant treatment of stage IIB and III melanoma, but adverse reactions such as neutropenia, liver toxicity, and fatigue cannot be ignored. In addition, according to research reports, continuous administration of interferon may induce CD8+T cell exhaustion, impair antitumor immunotherapy, and result in poor clinical antitumor efficacy of IFNα2b alone. Therefore, we currently need to explore a new immunotherapy approach based on IFNα2b, which has important guiding significance for improving the therapeutic effect of melanoma.

[0004] Oncolytic viruses are natural or genetically modified viruses that can selectively infect and kill tumor cells with little damage to normal cells. This type of virus mainly exerts its anti-tumor effect by directly lysing tumor cells and indirectly enhancing the host's anti-tumor immunity. Studies have shown that it has a synergistic effect when used in combination with chemotherapy, radiotherapy, PD1 and other treatment methods to treat tumors. At present, there are no relevant research reports on the combined use of oncolytic viruses and IFNα2b to treat melanoma in this field. Summary of the invention

[0005] The purpose of the present invention is to provide an application of an oncolytic adenovirus and IFNα2b in the preparation of a drug for treating melanoma, so as to solve the problems existing in the above-mentioned prior art. The present invention has found that the oncolytic adenovirus combined with IFNα2b has a significant synergistic tumor-suppressing effect in the treatment of melanoma, which has potential application prospects for the treatment of melanoma and can provide a more effective treatment plan for melanoma patients.

[0006] To achieve the above object, the present invention provides the following solutions:

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

[0008] The oncolytic adenovirus has a human telomerase promoter and an adenovirus replication initiation control E1A gene.

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

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

[0011] The human telomerase promoter hTERT replaced the promoter CMV in the shuttle plasmid pDC315 in 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, EcoR I and BamH I restriction sites were introduced into the primers to amplify the E1A gene fragment by PCR;

[0013] The EcoR I and BamH I double-digested fragment of the pDC315-hTERT-3E plasmid was connected 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, and after culture, purification was performed to obtain the oncolytic adenovirus.

[0015] The present invention also provides the use of the above-mentioned composition in preparing a drug for treating melanoma.

[0016] Furthermore, the composition plays a role in treating melanoma by inhibiting the growth of melanoma cells and promoting the apoptosis of melanoma cells.

[0017] The present invention also provides use of an oncolytic adenovirus in preparing a drug 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 comprises the following steps:

[0019] The human telomerase promoter hTERT replaced the promoter CMV in the shuttle plasmid pDC315 in 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, EcoR I and BamH I restriction sites were introduced into the primers to amplify the E1A gene fragment by PCR;

[0021] The EcoR I and BamH I double-digested fragment of the pDC315-hTERT-3E plasmid was connected 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, and after culture, purification was performed to obtain the oncolytic adenovirus.

[0023] The present invention also provides a drug for treating melanoma, wherein the active ingredient includes an oncolytic adenovirus or the above-mentioned combination;

[0024] The oncolytic adenovirus has a human telomerase promoter and an adenovirus replication initiation control E1A gene.

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

[0026] The human telomerase promoter hTERT replaced the promoter CMV in the shuttle plasmid pDC315 in 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, EcoR I and BamH I restriction sites were introduced into the primers to amplify the E1A gene fragment by PCR;

[0028] The EcoR I and BamH I double-digested fragment of the pDC315-hTERT-3E plasmid was connected 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, and after culture, purification was performed to obtain the oncolytic adenovirus.

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

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

[0032] The present invention proposes for the first time that oncolytic adenovirus combined with IFNα2b has a significant synergistic tumor-suppressing effect in the treatment of melanoma, which is specifically reflected in: in cell experiments, oncolytic adenovirus combined with IFNα2b can inhibit the growth of melanoma cells and promote the apoptosis of melanoma cells; in animal experiments, oncolytic adenovirus combined with IFNα2b can significantly inhibit the growth of in situ melanoma, promote the apoptosis of tumor cells, prolong the survival period and have good biocompatibility. The combination of oncolytic virus and IFNα2b has potential application prospects in the treatment of melanoma and can provide more effective treatment options for melanoma patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 Schematic diagram of the dosing regimen for B16-F10 tumor-bearing mice;

[0035] Figure 2 The Ki67 immunohistochemical staining image (A) and quantitative analysis result (B) of the tumor dissected after the experiment in Example 1;

[0036] Figure 3 The TUNEL immunohistochemical staining image (A) and quantitative analysis result (B) of the tumor dissected after the experiment in Example 1;

[0037] Figure 4 The flow cytometric images (A) and quantitative analysis results (B) of apoptosis of B16-F10 cells in different treatment groups in Example 2; n=3;

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

[0039] Figure 6 These are photos of tumors removed after the experiment in different treatment groups in Example 3; n=4;

[0040] Figure 7 This is a survival curve of B16-F10 tumor-bearing mice in different treatment groups in Example 3; n=5;

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

[0042] Fig. 9 These are H&E staining images of the main organs (heart, liver, spleen, lung, and kidney) of B10-F10 tumor-bearing mice in different treatment groups in Example 3. DETAILED DESCRIPTION

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

[0044] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0046] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

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

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

[0049] All quantitative experimental data were obtained by at least three independent replicates and presented as mean ± standard error (Mean ± SEM). GraphPad Prism (8.0.1) software was used for significance analysis by unpaired two-tailed t-test. 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 adenovirus (OA) referred to in the present invention is a selectively replicating adenovirus with a human telomerase promoter and an adenovirus replication initiation control E1A gene. By inserting a telomerase promoter sequence before the E1A gene that controls the initiation of adenovirus replication, an oncolytic adenovirus with tumor cell-specific replication ability was constructed (disclosed in Chinese patent CN201811026737.5), and the construction method is described in detail as follows:

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

[0052] a. The human telomerase promoter (hTERT) was used to replace the promoter CMV in the shuttle plasmid pDC315 in the recombinant adenovirus dual-plasmid system to construct the pDC315-hTERT-3E plasmid;

[0053] b. Using the adenovirus replication initiation control gene E1A cDNA fragment as a template, introducing EcoR I and BamHI restriction sites into the primers, and PCR amplifying E1A;

[0054] c. The EcoR I and BamH I double-digested fragment of plasmid pDC315-hTERT-3E was 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, the culture medium was changed every 2 days and the cell status was continuously observed;

[0057] 4) One week after transfection, cell plaques can be seen. When the cells become so cytopathic that they almost fall off the culture dish, collect the cells and culture supernatant.

[0058] 5) The oncolytic adenovirus was purified according to the operating procedures provided by the adenovirus purification kit Vivapure AdenoPACK 20RT, and the purified oncolytic adenovirus was stored at -80°C for future use.

[0059] After OA infects normal cells, due to the low telomerase content and activity in normal cells, the virus cannot start replication normally and is quickly cleared by the cells, causing no toxicity to the cells; however, when OA infects tumor cells, due to the presence of highly active telomerase in tumor cells, the virus is able to replicate rapidly and produce a large number of progeny viruses, which will lyse the infected tumor cells and kill them.

[0060] The oncolytic adenovirus culture method is as follows: take 1 mL of virus seed solution and inoculate it into a 10 cm culture dish with a monolayer of HEK293T cells and culture at 37°C and 5% CO. 2 Culture in the incubator for 1 hour, discard the original solution, add 10 mL of new DMEM virus maintenance solution containing 2% FBS, and incubate at 37°C and 5% CO 2 Continue to culture in the incubator. Observe daily. When all HEK293T cells show pathological changes, freeze and thaw three times in liquid nitrogen, centrifuge at 12000 rpm for 5 min, collect the supernatant and aliquot for use. Finally, 1×10 9 VPs / mL titer of oncolytic adenovirus.

[0061] The mouse melanoma cell line B16-F10 was cultured in a 10 cm cell culture dish containing 10% FBS, 1% penicillin-streptomycin (100 U / mL penicillin, 100 μg / mL streptomycin) and 1% glutamine RPMI 1640 medium at 37°C and 5% CO. 2 In a constant temperature incubator with a relative humidity of 95%, when the cell confluence reaches 80%, the medium is discarded, the cells are washed twice with PBS, and trypsin containing 0.25% EDTA is added to digest the cells for 1 min, and an equal volume of complete medium is added to terminate the digestion. The cell suspension is collected, centrifuged at 1000 rpm for 5 min, the supernatant is discarded, the cells are resuspended in complete medium, and the cells are passaged at a ratio of 1:5.

[0062] Example 1

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

[0064] B16-F10 tumor cells with a confluence of 80% were digested with trypsin containing 0.25% EDTA, centrifuged and discarded the supernatant, washed once with PBS, and resuspended in serum-free RPMI-1640 medium to adjust the cell density to 1×10 5 / mL, and 100 μL was inoculated into the back of C57BL / 6 mice to establish a tumor-bearing mouse model.

[0065] 2. Experimental Grouping and Treatment

[0066] The tumor-bearing mice were randomly divided into four groups, with 5 mice in each group. Figure 1 The dosage regimens shown were PBS, OA, IFNα2b, and IFNα2b+OA, where the dosage of OA was 1×10 8 PFU / mouse, the dosage of IFNα2b was 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 PBS, every other day, for two consecutive times;

[0068] (2) OA group: when the tumor volume reached 100 mm 3 Afterwards, each mouse was injected intratumorally with 1×10 8 PFU of OA, administered every other day for two consecutive times;

[0069] (3) IFNα2b group: when the tumor volume reached 100 mm 3 Afterwards, each mouse was injected subcutaneously with 1×10 4 U of IFNα2b, given every other day, twice in a row;

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

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

[0072] In order to further explore the differences in tumor growth in tumor-bearing mice after different treatments, after two consecutive administrations, the mice were killed by cervical dislocation, the tumors were dissected out, fixed in 4% paraformaldehyde for 24 h, made into 5 μm paraffin sections, and Ki67 and TUNEL immunohistochemical staining was performed as follows:

[0073] (1) Sequentially place the sections in 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 wash with distilled water for 5 min to dewax;

[0074] (2) The sections were immersed in Tris-EDTA antigen retrieval solution (pH = 9.0) and microwaved for 3 min at high heat and 15 min at low heat for antigen retrieval. The sections were then washed with PBS three times, 5 min each time.

[0075] (3) Immerse the slices in 3% H 2 O 2 The solution was incubated at room temperature in the dark for 30 min to inactivate endogenous peroxidase, and then washed with PBS three times, 5 min each time;

[0076] (4) Use an immunohistochemistry pen to draw a circle around the tissue, add the diluted primary antibody, incubate overnight at 4°C, and wash with PBS three times, 5 min each time;

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

[0078] (6) Add DAB colorimetric solution and rinse the sections with tap water to terminate color development;

[0079] (7) Hematoxylin staining for 5 min, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for 3 s, rinse with tap water, hematoxylin blueing for 10 s, rinse with tap water;

[0080] (8) The sections 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 make them transparent;

[0081] (9) After the slices are dried, they are sealed with neutral gum. Images are acquired using a digital pathology slice scanner to analyze tumor cell proliferation and apoptosis.

[0082] The results are as follows Figure 2 and Figure 3 Compared with the PBS group, the tumor tissues in the IFNα2b+OA group showed a large area of ​​cell apoptosis, enlarged intercellular spaces, condensed nuclei, and strong eosinophilic cytoplasm. The results of immunohistochemical experiments showed that the combination of IFNα2b and OA can effectively induce apoptosis and necrosis of tumor tissues, thereby significantly inhibiting tumor growth.

[0083] Example 2

[0084] B16-F10 cells with a confluence of 80% were digested with trypsin containing 0.25% EDTA, centrifuged and the supernatant was discarded. Tumor cells were diluted with complete medium and inoculated into 6-well cell culture plates, with 2×10 cells per well. 5 The cell culture plate was placed at 37°C and 5% CO 2 Culture overnight in a constant temperature incubator, discard the culture medium, wash twice with PBS, and divide the cells in the well plate into the following 4 groups according to n=3:

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

[0086] (2) OA group, treated with OA for 48 h;

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

[0088] (4) IFNα2b+OA group: mice were treated with OA and IFNα2b simultaneously for 48 h.

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

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

[0091] Depend on Figure 4 It can be seen that more than 90% of B16-F10 cells in the PBS group, OA group and IFNα2b group were Annexin V / PI double negative, which were normal cells; while 11% of B16-F10 cells in the IFNα2b+OA group were Annexin V / I double positive, which were late apoptotic cells, and another 14% of B16-F10 cells were Annexin V single positive, which were early apoptotic cells. The above results show that the combination of IFNα2b and OA can effectively promote the apoptosis of melanoma cells.

[0092] Example 3

[0093] B16-F10 melanoma efficacy evaluation

[0094] 1. Pharmacodynamic evaluation of tumor growth

[0095] A B16-F10 tumor-bearing mouse model was established by referring to the method in Example 1, and C57BL / 6 mice were randomly divided into 4 groups, with 5 mice in each group. Figure 1 The administration schedules were PBS, OA, IFNα2b, and IFNα2b+OA, with the OA dose of 1×10 8 PFU / mouse, the dosage of IFNα2b was 1×10 4 U / mouse. Starting from the first administration, the long diameter (L) and short diameter (W) of the mouse tumor were measured with an electronic vernier caliper every other day, and the tumor volume (V) was calculated according to the following formula and the tumor growth curve was drawn:

[0096] V=0.5×L×W 2 .

[0097] The results are as follows Figure 5 Compared with the PBS group, the administration of OA and IFNα2b alone had a certain anti-tumor effect, because they each had a certain regulatory effect on the tumor microenvironment and tumor tissue, and the IFNα2b+OA group could significantly inhibit tumor growth, achieving the effect of "1+1>2".

[0098] On the 12th day after administration, mice in each group were killed by cervical dislocation, and the tumors and major organs (heart, liver, spleen, lung, and kidney) were removed. Figure 6 Compared with other groups, the tumor volume of the IFNα2b+OA group was significantly reduced, which was consistent with the results of the tumor growth curve, showing a good immune combined therapy effect.

[0099] 2. Analysis of mouse survival

[0100] The grouping and dosing regimen of the B16-F10 melanoma model were the same as in 1. The long and short diameters of the mouse tumors were measured using an electronic vernier caliper to calculate the tumor volume. Once the tumor volume exceeded 1500 mm 3 , the mice were judged dead.

[0101] The survival results of B16-F10 tumor-bearing mice in different groups are as follows Figure 7 As shown, it can be found that on the 24th day after tumor bearing, all the mice in the PBS group died; the mice in the OA and IFNα2b groups began to die from the 9th day after tumor bearing; on the 27th day after tumor bearing, 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 from the first administration in Part 1, the body weight of mice in each group was measured every other day using an electronic balance, and a body weight change curve was drawn.

[0104] The weight changes of tumor-bearing mice during the experiment Figure 8 As shown, there was no significant decrease in body weight of mice in each group, indicating that the combined administration of IFNα2b+OA has good biosafety.

[0105] The main organs (heart, liver, spleen, lung, and kidney) of tumor-bearing mice in different treatment groups were fixed in 4% paraformaldehyde for 24 h, prepared into 5 μm paraffin sections, and the paraffin sections were stained with H&E as follows:

[0106] (1) Sequentially place the sections in 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 wash with distilled water for 5 min to dewax;

[0107] (2) The sections were stained with hematoxylin for 10 min and rinsed with distilled water;

[0108] (3) The sections were stained in eosin solution for 3 min and then rinsed with distilled water;

[0109] (4) The sections 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 make them transparent;

[0110] (5) After the slices are dried, they are sealed with neutral gum. Images are acquired using a digital pathology slice scanner to observe the microscopic morphology of the tissue.

[0111] The results of H&E staining of the main organs of mice in each group are shown in Fig. 9 As shown, no obvious inflammation or microstructural damage was observed in the heart, liver, spleen, lung and kidney of tumor-bearing mice after drug treatment, further confirming that IFNα2b+OA has good biocompatibility.

[0112] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A composition for treating melanoma, characterized in that: These include oncolytic adenovirus and IFNα2b; The oncolytic adenovirus has a human telomerase promoter and an adenovirus replication initiation control E1A gene.

2. The composition according to claim 1, characterized in that The ratio of the oncolytic adenovirus to the IFNα2b is 10 8 PFU: 10 4 U.

3. The composition according to claim 1, characterized in that The method for constructing the oncolytic adenovirus comprises the following steps: The human telomerase promoter hTERT replaced the promoter CMV in the shuttle plasmid pDC315 in the recombinant adenovirus dual-plasmid system to construct the pDC315-hTERT-3E plasmid; Using the adenovirus replication initiation control gene E1A cDNA fragment as a template, EcoR I and BamH I restriction sites were introduced into the primers to amplify the E1A gene fragment by PCR; The EcoR I and BamH I double-digested fragment of the pDC315-hTERT-3E plasmid was connected with the E1A gene fragment 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, and after culture, purification was performed to obtain the oncolytic adenovirus.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a drug for treating melanoma.

5. The use according to claim 4, characterized in that: The composition plays a role in treating melanoma by inhibiting the growth of melanoma cells and promoting the apoptosis of melanoma cells.

6. Use of an oncolytic adenovirus in the preparation of a drug for treating melanoma, characterized in that: The oncolytic adenovirus has a human telomerase promoter and an adenovirus replication initiation control E1A gene.

7. The use according to claim 6, characterized in that: The method for constructing the oncolytic adenovirus comprises the following steps: The human telomerase promoter hTERT replaced the promoter CMV in the shuttle plasmid pDC315 in the recombinant adenovirus dual-plasmid system to construct the pDC315-hTERT-3E plasmid; Using the adenovirus replication initiation control gene E1A cDNA fragment as a template, EcoR I and BamH I restriction sites were introduced into the primers to amplify the E1A gene fragment by PCR; The EcoR I and BamH I double-digested fragment of the pDC315-hTERT-3E plasmid was connected with the E1A gene fragment 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, and after culture, purification was performed to obtain the oncolytic adenovirus.

8. A drug for treating melanoma, characterized in that: The active ingredient comprises an oncolytic adenovirus or a composition according to any one of claims 1 to 3; The oncolytic adenovirus has a human telomerase promoter and an adenovirus replication initiation control E1A gene.

9. The drug according to claim 8, characterized in that The method for constructing the oncolytic adenovirus comprises the following steps: The human telomerase promoter hTERT replaced the promoter CMV in the shuttle plasmid pDC315 in the recombinant adenovirus dual-plasmid system to construct the pDC315-hTERT-3E plasmid; Using the adenovirus replication initiation control gene E1A cDNA fragment as a template, EcoR I and BamH I restriction sites were introduced into the primers to amplify the E1A gene fragment by PCR; The EcoR I and BamH I double-digested fragment of the pDC315-hTERT-3E plasmid was connected with the E1A gene fragment 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, and after culture, purification was performed to obtain the oncolytic adenovirus.

10. The drug according to claim 8, characterized in that The drug also includes pharmaceutically acceptable excipients.

Images