A replicating oncolytic adenovirus capable of inhibiting tumor progression and prolonging the survival time of tumor-bearing individuals and its application

Abstract

The present invention relates to the field of tumor biotherapy, and specifically to a replicating oncolytic adenovirus Ad5 GLP1 that can inhibit tumor progression and prolong the survival time of tumor-bearing individuals and its application. The present invention discloses a design and construction method of Ad5 GLP1, and successfully obtains a replicating oncolytic adenovirus Ad5 GLP1, which can specifically replicate in tumor cells and secrete to the outside of the cells, can highly express the glucagon-like peptide-1 receptor agonist-glucagon-like peptide 1 (GLP1), and the protein molecules can be secreted to the outside of the cells in large quantities and can significantly promote viral replication. Experiments show that the novel replicating oncolytic adenovirus of the present invention has the effects of significantly enhancing anti-tumor immunity, inhibiting the growth of solid tumors, and prolonging the survival of tumor-bearing individuals, and has great prospects and value for the development of anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of tumor biological treatment, and in particular to a replicating oncolytic adenovirus capable of inhibiting tumor progression and prolonging the survival time of tumor-bearing individuals and an application thereof. Background Art

[0002] Cancer has become the leading disease that threatens human life and health. Surgery is the most effective treatment for early-stage cancer, but it does not significantly improve the survival of most patients with advanced and metastatic disease. Although radiotherapy, chemotherapy and other adjuvant treatments can inhibit tumor progression to a certain extent, the overall survival rate of cancer patients has not been significantly improved. Finding new, safe and effective therapeutic drugs is a hot topic in pharmaceutical research around the world today.

[0003] In recent years, tumor immunotherapy has attracted much attention due to its remarkable efficacy. Tumor immunotherapy is a treatment method that activates the body's immune system to kill and monitor cancer cells by activating and / or regulating immune-related pathways. As a rising star in the field of tumor immunotherapy, oncolytic virus has received increasing attention with the approval of the oncolytic virus T-Vec by the FDA at the end of 2015. Oncolytic viruses are a type of virus that has the ability to replicate itself and replicate in large quantities in tumor cells. They have a variety of unique anti-tumor effects: they can induce immunogenic death of tumor cells; activate the body's immune surveillance; and express recombinant proteins to regulate the tumor microenvironment. Currently, there are many clinical trials related to oncolytic viruses underway, including adenovirus, herpes simplex, measles virus, vaccinia virus, herpes stomatitis virus, etc., and good therapeutic effects have been achieved.

[0004] However, solid tumors escape the body's immune surveillance and promote invasion and metastasis through a variety of mechanisms, including abnormal metabolism and pro-inflammatory microenvironment. Glucagon-like peptide 1 (GLP1) is a 30-amino acid peptide, a glucagon-like peptide-1 receptor agonist that not only lowers blood sugar, but also significantly inhibits multiple tumor-promoting inflammatory pathways, including the activation of the NF-κB / IL-6 / STAT3 pathway and the production of NLRP3 / IL-1β inflammasomes.

[0005] Studies have found that while oncolytic viruses activate immunity, they also upregulate pro-tumor inflammation in the tumor microenvironment and change the metabolic microenvironment, which is not conducive to their anti-tumor effects. How to effectively overcome the inherent deficiencies in oncolytic virus therapy is an important scientific problem that the present invention aims to solve. Summary of the invention

[0006] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a replicative oncolytic adenovirus and its application that can inhibit tumor progression and prolong the survival time of tumor-bearing individuals; the oncolytic adenovirus of the present invention can infect tumor cells and replicate and secrete outside the cells, and the glucagon-like peptide-1 receptor agonist expressed by the virus can be secreted outside the cells and promote viral replication, inhibit tumor-promoting inflammation, and can significantly inhibit tumor growth; finally, the invention establishes a variety of tumor models, including liver cancer, pancreatic cancer, etc., to verify the significant effect of oncolytic adenovirus in treating tumors and prolonging survival.

[0007] The technical solution adopted by the present invention to solve the technical problem is:

[0008] A replicating oncolytic adenovirus capable of inhibiting tumor progression and prolonging the survival of tumor-bearing individuals, the oncolytic adenovirus comprising a nucleic acid encoding a glucagon-like peptide-1 receptor agonist.

[0009] Among them, the oncolytic adenovirus can selectively replicate and dissolve tumors in tumors, and can also replicate and express glucagon-like peptide-1 receptor agonist, a protein that can regulate tumor microenvironment metabolism and inhibit tumor-promoting inflammation.

[0010] Preferably, the amino acid sequence of the glucagon-like peptide-1 receptor agonist is selected from the following (1) or (2):

[0011] (1) the amino acid sequence as described in SEQ ID NO: 2;

[0012] (2) An amino acid sequence that has more than 80% homology with the sequence described in SEQ ID NO: 2 and is associated with oncolytic activity.

[0013] The glucagon-like peptide-1 receptor agonist is a soluble protein GLP1, which can promote the replication of oncolytic adenovirus.

[0014] The replicative oncolytic adenovirus is Ad5 GLP1, which is a type 5 recombinant adenovirus.

[0015] The present invention also protects the use of the above-mentioned replicative oncolytic adenovirus in the preparation of anti-tumor drugs.

[0016] Preferably, the tumor is one of liver cancer, pancreatic cancer and colon cancer.

[0017] The present invention also protects a pharmaceutical composition, which comprises the replicative oncolytic adenovirus described above, and a pharmaceutically acceptable excipient.

[0018] A method for constructing a replicative oncolytic adenovirus that inhibits tumor progression and prolongs the survival time of tumor-bearing individuals is achieved through three steps: plasmid construction, virus rescue and virus amplification.

[0019] Preferably, the method comprises the following specific steps:

[0020] Step 1, plasmid construction: linearize the constructed shuttle vector pShuttle-GLP1 with PmeI and transfer it into competent medium pAdEasy-BJ5183, screen positive clones for culture and identification, and re-transform the correct clone plasmid into DH5a competent medium for secondary screening and identification. After the correct identification, perform large-scale plasmid extraction to obtain the full-length plasmid of Ad5 GLP1;

[0021] Step 2, virus rescue: Ad5 GLP1 full-length plasmid was linearized with PacI, purified and transfected into 293T cells until 80% of the cells showed pathological changes, the cells were blown off with culture medium and collected into centrifuge tubes, repeatedly frozen and thawed and centrifuged, and the virus supernatant was collected and stored at -80°C as virus seed;

[0022] Step 3, virus amplification: Take the virus seed solution and add it to the 293T cell plate for culture. When the cell density reaches more than 90%, subculture at a ratio of 1 to 3 until 80% of the cells show pathological changes. Collect the virus according to the method in step 2 and purify the virus by centrifugation.

[0023] More preferably, the shuttle vector Ad5-pShuttle-GLP1 is constructed as follows: first, the DNA sequence of GLP1-E1A is synthesized, as shown in SEQ ID NO: 1; the aforementioned sequence is double-digested with BamHI and XhoI, and the pShuttle plasmid is also double-digested with BamHI and XhoI; the digested DNA and the pShuttle plasmid are ligated with T4 ligase; the ligation product is transformed into DH5a competent cells, and positive clones are screened on kanamycin-resistant LB plates; positive monoclonal DH5a bacteria are amplified, and plasmid extraction is performed; this plasmid is the shuttle-GLP1 plasmid used to prepare the pAd5 GLP1 adenovirus.

[0024] The oncolytic adenovirus constructed by the above construction method is Ad5 GLP1.

[0025] The present invention also protects the use of the soluble protein GLP1 in the preparation of drugs for promoting oncolytic adenovirus replication and oncolytic drugs.

[0026] The present invention also protects the use of the soluble protein GLP1 in the preparation of anti-tumor drugs.

[0027] The present invention also protects the use of the soluble protein GLP1 in preparing drugs for inhibiting inflammatory pathways.

[0028] Beneficial Effects

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention discloses a method for constructing a replicative oncolytic adenovirus Ad5 GLP1. After Ad5 GLP1 infects cells, it can replicate and express the target protein GLP1 in the cells.

[0031] (2) GLP1 protein can significantly promote the replication of oncolytic adenovirus in tumor cells.

[0032] (3) GLP1 protein can significantly promote the oncolytic effect of oncolytic adenovirus.

[0033] (4) GLP1 protein can significantly inhibit the activation of tumor-promoting inflammatory pathways induced by inflammatory factors and oncolytic adenovirus.

[0034] (5) Ad5 GLP1 can significantly inhibit the activation of tumor-promoting inflammatory pathways induced by inflammatory factors and oncolytic adenovirus.

[0035] (6) Ad5 GLP1 can significantly promote the anti-tumor immune response induced by oncolytic adenovirus.

[0036] (7) In animal experiments, Ad5 GLP1 significantly inhibited the growth of subcutaneous liver cancer tumors and significantly prolonged the survival time of mice.

[0037] (8) In animal experiments, Ad5 GLP1 significantly inhibited the growth of pancreatic cancer and significantly prolonged the survival time of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The results of the present invention show the construction strategy of the replicative oncolytic adenovirus Ad5 GLP1; wherein A is a schematic diagram of the construction method of the Ad5GLP1 virus; B is the PCR result of the target gene GLP1, wherein 1 is the PCR product and 2 is the DL2000DNA Maker.

[0039] Figure 2 (A) LM3 human liver cancer cells and (B) Hepa1-6 mouse liver cancer cells were infected with the recombinant oncolytic adenoviruses Ad5 GLP1 and Ad5 con (MOI = 5) expressing the soluble protein GLP1 of the present invention, respectively. After 24 hours, the expression of green fluorescent protein was observed under a fluorescence microscope, and then the infected cells were collected and the expression of the target protein GLP1 was detected by WB. The data represent three independent repeated experiments. Green fluorescent protein expression can show viral replication.

[0040] Figure 3The effect of the soluble protein GLP1 of the present invention on the replication of oncolytic adenovirus, A is 48 hours, B is 72 hours. Liver cancer LM3 cells were inoculated into a 96-well culture plate, and the cells were treated with adenovirus expressing luciferase (MOI=5) with or without GLP1 (20 μM) and then continued to be cultured. Luciferase substrate was added after 48 and 72 hours, and the fluorescence intensity was detected.

[0041] Figure 4 The present invention is a comparison of the replication ability of Ad5 GLP1 in tumor cells with the control virus Ad5 con. Colon cancer MC38 cells were infected with Ad5 con and Ad5 GLP1 at an MOI of 1, and the cells were collected and total RNA was extracted at 12, 24, 48, 60 and 72 hours, and the gene expression level of the virus-specific protein Hexon was detected by quantitative RT-PCR. The data were expressed as 1 at 12 hours, showing the multiple increase in virus replication.

[0042] Figure 5 It is the effect of the secretory protein GLP1 of the present invention on the oncolytic effect of oncolytic adenovirus; wherein, A is the effect on cell activity, and B is the effect on the number of viable cells. HCC-LM3 liver cancer cells were seeded in 96-well plates or 6-well plates. After they adhered to the wall, adenovirus with an MOI value of 5 was used to infect liver cancer cells HCC-LM3, with or without GLP1 (20 μM). The untreated group and the GLP1-treated group were used as controls. After 48 hours of culture, the proportion of viable cells was detected by MTT method or the number of viable cells after trypan blue staining was detected using CountStar counter. The results are mean (Mean) + standard deviation (SD), compared with the control group: #P>0.05; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001.

[0043] Figure 6The soluble GLP1 of the present invention significantly inhibits the activation of inflammatory pathways induced by inflammatory factors or oncolytic adenovirus. Verification of the biological function of the anti-inflammatory peptide genetic engineering plasmid GLP1 A. Human or mouse liver cancer cells were inoculated in a 6-well plate, and after adherence, GLP1 plasmid or blank plasmid was transfected respectively. After 24 hours, IL-6 (25ng / ml) was added to stimulate for 0.5 hours, and cell protein was collected. The phosphorylation level of STAT3 was detected by Western Blot. B. Human liver cancer cells HCC-LM3 were inoculated in a 6-well plate, and after adherence, GLP1 plasmid or blank plasmid was transfected respectively. After 24 hours, adenovirus (MOI=5) was added. After co-culture for 24 hours, cell RNA was extracted and reversely transcribed into cDNA. The mRNA levels of the corresponding inflammatory factors TNF-α, IL-6 and IL-1β were detected by qRT-PCR. The results are mean (Mean) + standard deviation (SD), compared with the control group: #P>0.05; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001.

[0044] Figure 7 The recombinant oncolytic adenovirus Ad5 GLP1 of the present invention significantly inhibits the activation of the virus-induced tumor-promoting inflammatory pathway, wherein A corresponds to HCC-LM3 cells and B corresponds to Hepa1-6 cells. HCC-LM3 and Hepa1-6 liver cancer cells were inoculated in 6-well plates, respectively. After the cells adhered to the wall, they were infected with recombinant oncolytic adenovirus (MOI = 5) for 24 hours, and then the cells were collected and proteins and RNA were extracted respectively. The levels of corresponding inflammatory factors were detected by western blot and qRT-PCR. The results are mean (Mean) + standard deviation (SD), compared with the control group: #P>0.05; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001.

[0045] Figure 8 The recombinant oncolytic adenovirus AD5 GLP1 of the present invention has a significant inhibitory effect on the activation of the pathway induced by the inflammatory factor IL-6. A and B. HCC-LM3 and Hepa1-6 cells were plated, and after 24 hours, IL-6 (25ng / ml) was added to the culture medium to stimulate for 0.5 hours, and the cell proteins were collected, and the phosphorylation levels of STAT3 and IκBα were detected by western blot.

[0046] Fig. 9The secretory protein GLP1 of the present invention enhances the anti-tumor immune response of the recombinant oncolytic adenovirus. A mouse subcutaneous transplant tumor model was constructed with mouse liver cancer Hepa1-6 cells. When the mouse tumor grew to 0.4 cm×0.4 cm, the mice were randomly divided into 4 groups. On the second day, the treatment group was given GLP1 (300 μg / kg / mouse) intratumoral injection, and the adenovirus Ad5 treatment group was given adenovirus 5×10 8 PFU / mouse, intratumoral injection, once a day. The combined treatment group was given GLP1 and adenovirus treatment at the same time, and the control group was given the same volume of PBS intratumoral injection. One week later, the mice were killed by cervical dislocation, and the tumor tissue was separated, chopped and digested with collagenase, then passed through a cell sieve to form a single cell suspension, and the ELISpot method was used to detect the amount and intensity of IFN-γ released by immune cells.

[0047] Fig.10 The recombinant oncolytic adenovirus Ad5 GLP1 of the present invention has a significant inhibitory effect on the growth of liver cancer. Treatment of hepatocellular carcinoma. A. Flow chart of the in vivo experimental scheme. Mice were inoculated with 1×10 7 / HCC-LM3 cells, the tumor size and body weight of mice were measured for the first time on day 0, and the mice were randomly divided into 3 groups, including control group, Ad5 GLP1 group and Ad5 con group, with 6-7 mice in each group. The treatment group was injected with Ad5 GLP1 intratumorally, 1×10 9 PFU / mouse, or the same dose of Ad5 con, the control group mice were injected with the same volume of PBS, and adenovirus was injected once every 5 days until the tumor volume of the mice was greater than 2.0cm 3 Or the mice died. B. Adenovirus injection started on day 0, and the tumor size was measured every two days. The figure shows the tumor size of mice in each group. C. Adenovirus injection started on day 0, and the mice were weighed every two days. The figure shows the weight of mice in each group. The results are mean (Mean) + standard deviation (SD), #P>0.05; *P<0.05; **P<0.01.

[0048] Fig.11 The recombinant oncolytic adenovirus Ad5 GLP1 of the present invention can significantly prolong the survival of mice bearing human LM3 liver cancer. The in vivo experiment was divided into 3 groups, including a control group, a recombinant adenovirus Ad5 GLP1 group and a control adenovirus Ad5 con group, with 6-7 mice in each group. When the mouse tumor was larger than 2.0 cm 3 Or death due to tumor burden was regarded as death case, and the survival time of mice was counted. The results are mean (Mean) + standard deviation (SD), compared with the control group: #P>0.05; *P<0.05; **P<0.01.

[0049] Fig.12The recombinant oncolytic adenovirus Ad5 GLP1 of the present invention can significantly inhibit the growth of liver cancer in immune-competent mice without obvious toxic side effects. Balbc male mice were inoculated with 5x10 6 H22 liver cancer cells, when the tumor grows to about 5x7mm, 3x10 8 pfu Ad5 GLP1, once every other day for a total of three times, and the tumor diameter (left) and mouse body weight changes (right) were measured every other day.

[0050] Fig.13 The recombinant oncolytic adenovirus Ad5 GLP1 of the present invention can significantly inhibit the growth of pancreatic cancer in immunocompetent mice without obvious toxicity. Balbc male mice were inoculated with 5x10 6 Panc02 pancreatic cancer cells, when the tumor grows to about 5x7mm, 3x10 8 pfu Ad5 GLP1, once every other day for a total of three times, and the tumor diameter (left) and mouse body weight changes (right) were measured every other day.

[0051] Fig.14 The recombinant oncolytic adenovirus Ad5 GLP1 of the present invention can significantly prolong the survival time of pancreatic cancer-bearing mice, and 33% of the mice were cured of tumors and survived for a long time. Balbc male mice were inoculated with 5x10 6 Panc02 pancreatic cancer cells, when the tumor grows to about 5x7mm, 3x10 8 pfu Ad5 GLP1, once every other day for a total of three times, and the survival time of the mice was monitored. DETAILED DESCRIPTION

[0052] The present invention is further explained and illustrated below in conjunction with specific examples, but it should be understood that the examples given are only for illustration and do not constitute any limitation to the present invention in any way.

[0053] 1 Experimental Materials and Methods

[0054] 1.1 Experimental Materials and Instruments

[0055] 1.1.1 Experimental cell lines

[0056] Human embryonic kidney cell line 293T, human liver cancer cell line HCC-LM3, mouse liver cancer cell lines Hepa1-6 and H22, mouse pancreatic cancer cell line Panc02, and mouse colon cancer cell line MC38 were cultured in a high-glucose DMEM medium containing 10% fetal bovine serum, 100U / I penicillin and 1mg / ml streptomycin at 37°C in a 5% CO2 incubator.

[0057] 1.1.2 Experimental instruments

[0058] Biological Safety Cabinet ( III advance, Class II Biological Safety Cabinet, The Baker Company), CO2 incubator (FORMA SERIES II WATER JACKET CO2incubator, Thermo), low-temperature centrifuge (HERAEUS MEGAFUGE 1.0R, Thermo), vertical electrophoresis tank (BIO-RAD), electrophoresis apparatus (BIO-RAD), semi-dry transfer-transfer apparatus (BIO-RAD), immunoblotting exposure system (Alpha Innotech), PCR instrument (PCRThermal Cycler Dice, TaKaRa), real-time quantitative PCR instrument and analysis software (ABI384, Sequence DetectionSoftware, Version 1.3.1), microplate reader (VERSA max microplate reader), complete set of pipettes (eppendorf and RAININ), cell counter (Countstar Automated cell counter, Inno-AllianceBiotech Inc., Wilmington, USA), flow cytometer (FACSCalibur, Becton, Dickinson and Company, USA), FlowJo software (Version 7.6.5, Tree Star Inc, Ashland, Oregon), microplate oscillator (QiLinBeiEr), nucleic acid purity and concentration detector (Biophotometer plus, eppendorf), digital display constant temperature water bath (Guohua Electric).

[0059] 1.1.3 Main experimental reagents and consumables

[0060] Primers were synthesized by GenScript. DMEM high-glucose medium, double antibodies, and serum required for tumor cell culture were purchased from Invitrogen (Shanghai). Quantitative RT-PCR reagent Faststart Universal SYBR Green Master (Roche, 04913914001). Reagents and consumables required for Western Blot: protease inhibitor (Roche, 11873580001), cell lysis buffer (Biyuntian: P0013), PVDF membrane (Roche, 03010040001), WB Immobilon ECL luminescent solution (Millipore, WBKLS0500), primary antibody diluent (Biyuntian, P0023A), HRP-labeled secondary antibody (Multisciences, GAR007 and GAM007, 1:5000 dilution), and other required reagents were all domestic analytical grade and purchased from the School of Chemistry and Chemical Engineering of Nanjing University. Trypan blue (Biyuntian, C0011), Opti-MEM were purchased from Invitrogen (Shanghai) Company. Western Blot antibodies: anti-GAPDH (Bioworld, MB001, 1:5000 dilution), anti-p-STAT3 (Cell Signaling Technology, 4814S, 1:1000 dilution), anti-p-IκBα (Cell Signaling Technology, 2859S, 1:1000 dilution), anti-STAT3 (Cell Signaling Technology, 9139S, 1:1000 dilution), anti-IκBα (Cell Signaling Technology, 4814S, 1:1000 dilution). HRP anti-mouse IgG (Abbkine, A21010-1, 1:1000 dilution), HRP anti-rabbit IgG (Abbkine, A21020-1, 1:1000 dilution).

[0061] 1.2 Experimental methods

[0062] 1.2.1 Ad5 GLP1 virus construction (plasmid construction, virus rescue and amplification)

[0063] A. Construction of full-length Ad5 GLP1 plasmid:

[0064] First, the DNA sequence of GLP1-E1A is synthesized, as shown in SEQ ID NO: 1; the aforementioned sequence is double-digested with BamHI and XhoI, and the pShuttle plasmid is also double-digested with BamHI and XhoI; the digested DNA and the pShuttle plasmid are connected with T4 ligase; the ligation product is transformed into DH5a competent cells, and positive clones are screened on kanamycin-resistant LB plates; positive monoclonal DH5a bacteria are amplified, and plasmids are extracted. This plasmid is the shuttle-GLP1 plasmid used to prepare pAd5 GLP1 adenovirus.

[0065] The constructed plasmid pShuttle-GLP1 was linearized with PmeI and then transferred into the competent medium pAdEasy-BJ5183. The plasmid was screened using LB plates containing 50ug / ml kanamycin. The positive clones were picked for culture and identification. The correct clone plasmid was re-transformed into the DH5a competent medium for secondary screening and identification. After the correct identification, the plasmid was extracted to obtain the full-length plasmid of Ad5 GLP1.

[0066] B. Ad5 GLP1 virus rescue:

[0067] The full-length plasmid of Ad5 GLP1 was linearized using PacI. After purification, 1ug / well was added to a 6-well plate to transfect 293T cells and cultured at 37°C with 5% CO2. After 2 days, the cells were digested and transferred to a 10 cm dish. The medium was changed every 2-3 days until 80% of the cells showed pathological changes. The cells were blown off using 10 ml of culture medium and collected in a 15 ml centrifuge tube. The tubes were frozen and thawed twice, and centrifuged at 3000 rpm / min for 15 min. The viral supernatant was collected and stored at -80°C as a virus seed.

[0068] C. Virus Amplification:

[0069] Take 50ul of virus seed solution and add it to 60% 293T cells in a 10cm dish. Culture at 37℃ with 5% CO2 until the cell density reaches above 90%. Subculture at a ratio of 1 to 3 until 80% of the cells show pathological changes and there are about 10 dishes of cells. Collect the virus according to the above method and purify the virus by cesium chloride density gradient centrifugation; use TCID50 method for titer determination.

[0070] Functional evaluation of Ad5 GLP1 virus

[0071] A. Expression and secretion function of GLP1:

[0072] After 72 hours of infection of tumor cells with Ad5 GLP1 virus, cells and supernatant were collected and the expression and secretion function of GLP1 were detected by WB.

[0073] B. Virus replication ability:

[0074] Tumor cells were infected with Ad5 GLP1 and Ad5 con (Ad5 con is adenovirus type 5 that only contains the viral replication element E1A sequence but not the GLP1 sequence) at the same MOI. The cells were harvested at different times, and equal amounts of virus suspensions were obtained after repeated freeze-thaw and centrifugation. 293T cells were used to measure the virus titer and analyze changes in virus replication ability.

[0075] C. Oncolytic function:

[0076] Tumor cells were infected with Ad5 GLP1 and Ad5 con viruses at MOIs of 1 to 100, respectively. Cell activity was detected using MTT 72 hours later to evaluate the tumoricidal effect of Ad5 GLP1.

[0077] 1.2.2 In vivo study of the anti-tumor effects and mechanisms of Ad5 GLP1

[0078] A. Use 6-8 week old Balb / c or C57BL / 6 mice to establish subcutaneous tumor model in the right axilla or subcutaneous area. After 4-6 days, measure the tumor size to 200 mm. 3 The mice were randomly divided into three groups: no treatment group, control Ad5 con virus treatment group, and Ad5 GLP1 virus treatment group. The corresponding viruses were injected intratumorally according to the groups, and the amount of virus injected per mouse was 2.5×10 8 pfu, and followed up to measure the tumor volume and body weight. After the mice died naturally, the survival time of the mice was recorded.

[0079] 1.2.3 Ad5 GLP1 virus titer determination

[0080] 1) 293T cells were seeded in 96-well plates, with approximately 1×10 cells per well. 3 After the cells adhered to the wall, the titer was determined.

[0081] 2) Virus gradient dilution: prepare EP tubes, add 1170 μl of DMEM containing fetal bovine serum to each EP tube; add 130 μl of virus solution to the first EP tube, mix well, and mark as 10-1; draw 50 μl from the first EP tube into the second EP tube, mix well, and mark as 10-2; and so on, until the required gradient is reached.

[0082] 3) Add 100 μl of the corresponding gradient virus dilution solution to each well, repeat 10 wells for each gradient, and culture at 37°C overnight.

[0083] 4) After 5 days, place the 96-well plate under a microscope to observe GFP and record the number of wells with GFP in each gradient for calculation of virus titer.

[0084] 5) Calculation formula for virus titer TCID50:

[0085] Log10(TCID 50 )=L+d(s-0.5)+log10(1 / v);

[0086] Wherein, L = Log10 highest dilution (e.g., the highest dilution is 10-fold dilution, L = 1);

[0087] V = initial volume of cell culture medium per well (ml / well);

[0088] d = Log10 dilution (e.g. 10-fold dilution, d = 1);

[0089] s = sum of GFP ratios of each gradient.

[0090] 1.2.4 Quantitative PCR

[0091] The 10 μl system of real-time quantitative PCR consists of: 2.6 μl PCR water, 0.2 μl of upstream and downstream primers, 2 μl of template and 5 μl of SYBR Green fluorescent dye. After the samples are mixed, amplification is performed on an ABI 384 PCR instrument.

[0092] 1.2.5 Extraction and concentration determination of total cell protein

[0093] 1) Taking a six-well plate as an example, remove the cell culture supernatant, wash twice with PBS, remove the PBS, add 200 μl of trypsin to each well, digest and blow the cells, and collect the cells into an EP tube, and centrifuge at 1500 rpm for 5 minutes.

[0094] 2) Remove the supernatant, add PBS to resuspend the cells, and centrifuge at 1500 rpm for 5 minutes.

[0095] 3) Remove PBS, add the corresponding cell lysis solution containing protease inhibitors to each well according to the cell volume, vortex for 30 seconds, place on ice for 10 minutes, repeat the operation three times. Centrifuge at 12000g for 15 minutes at 4℃. Collect the supernatant in another clean EP tube.

[0096] 4) Determination of protein concentration: Detection was performed according to the instructions of the BCA protein concentration assay kit. Take 2 μl of protein sample in a 96-well plate, add 18 μl of PBS to dilute the sample, and finally add 200 μl of the assay working solution (the working solution is reagent A: reagent B = 50:1), place in a 60°C oven, and after 30 minutes, use an enzyme reader to measure the absorbance at 562 nm, and calculate the concentration of the protein sample according to the standard curve.

[0097] 5) Add 1 / 4 of the volume of protein lysate to each tube with 5× loading buffer, mix well, place in a 100℃ metal bath for 5 min, cool, and store at -20℃ for later use.

[0098] 1.2.6 Western blot experiment

[0099] 1) Gel preparation and electrophoresis: Prepare SDS-PAGE separation gel and stacking gel of different concentrations according to different requirements. According to the calculation results of protein quantification, the loading amount of each sample is adjusted to 30μg. Electrophoresis conditions: stacking gel 80V 30min, separation gel 120V, about 80min, provided that the bands are separated and do not run away.

[0100] 2) Transfer: Prepare filter paper and PVDF membrane, soak the PVDF membrane with methanol first, and then soak it in transfer buffer together with the filter paper for later use. Carefully remove the gel from the glass plate, soak it in transfer buffer, and place it in the order of negative electrode-filter paper-PVDF membrane-gel-filter paper-positive electrode, drive away bubbles, and transfer the membrane at a constant current of 110mA for 60-70min depending on the required strip size.

[0101] 3) Blocking: After the transfer, take out the PVDF membrane immediately and put it into 5% skim milk powder for blocking at room temperature for 1 hour.

[0102] 4) Primary antibody incubation: Incubate the cells with primary antibody at 4°C overnight.

[0103] 5) Secondary antibody incubation: Wash the strips with washing buffer for 10 min each time, for a total of three times; then incubate with the corresponding HPR-labeled secondary antibody at room temperature for 1 h.

[0104] 6) Exposure: Wash the strips with washing buffer for 10 min each time, for a total of three times; expose them on a WB exposure instrument with chemiluminescent solution and obtain the strip image.

[0105] 1.2.7 Trypan blue counting

[0106] Take a six-well plate as an example. Remove the cell supernatant, wash twice with PBS, remove the PBS, add 200μl trypsin to each well, gently blow the cells and collect them into a clean EP tube, centrifuge at 1500rpm for 5min. Remove the supernatant, add PBS to resuspend the cells, centrifuge at 1500rpm for 5min. Remove the PBS, add a certain amount of PBS to resuspend the cells according to the number of cells, take out 10μl of the cell resuspension, add 10μl of 0.2% trypan blue solution and mix, take 20μl of the mixture in a cell counting plate, and count with a cell counter.

[0107] 2. Results and Conclusions

[0108] See also Figure 2 The results showed that the replicative oncolytic adenovirus Ad5 GLP1 we constructed had the same ability to infect tumor cells and express the target gene as the control virus. In human and mouse liver cancer cell lines HCC-LM3 and Hepa1-6 cell lines, Ad5 GLP1 was able to efficiently infect cells and express GLP1 protein.

[0109] See also Figure 3 The results showed that soluble GLP1 can significantly promote the replication of oncolytic adenovirus in tumor cells. In the culture medium containing GLP1 protein, the replication of oncolytic adenovirus (Ad5) in liver cancer cells (LM3) increased significantly, indicating that GLP1 has the effect of promoting the replication of oncolytic adenovirus.

[0110] See also Figure 4 The results showed that the replication of the recombinant oncolytic adenovirus Ad5 GLP1 in tumor cells was significantly higher than that of the control oncolytic adenovirus. In mouse colon cancer cells, the mouse colon cancer cell line (MC38) was infected with Ad5 GLP1 at an MOI of 1. The gene expression number of Hexon of Ad5 GLP1 was significantly higher than the copy number of the control virus 48, 60 and 72 hours after the cells were infected, indicating that the replication ability of Ad5 GLP1 in tumors was significantly enhanced.

[0111] See also Figure 5 The results showed that soluble GLP1 can promote the oncolytic effect of adenovirus. In the culture medium containing GLP1 protein, the oncolytic effect of adenovirus on liver cancer cells (LM3) was significantly higher than that of the virus or GLP1 alone treatment group, indicating that GLP1 promotes adenovirus oncolysis.

[0112] See also Figure 6 The results showed that GLP1 can significantly inhibit the activation of inflammatory pathways caused by inflammatory factors or oncolytic viruses. In human or mouse liver cancer cell lines, GLP1 can inhibit the activation of the STAT3 pathway by the inflammatory factor IL-6, and significantly reduce the expression of virus-induced inflammatory factors, including tumor necrosis factor (TNF-α), interleukin 6 (IL-6) and interleukin 1 (IL-1β).

[0113] See also Figure 7 The results showed that the recombinant Ad5 GLP1 adenovirus can significantly inhibit the activation of virus-induced tumor-promoting inflammatory pathways. In human and mouse liver cancer cell lines, the TNF-α / NFkB and IL-6 / STAT3 pathways were significantly inhibited in the Ad5 GLP1-treated group.

[0114] See also Figure 8The results showed that the recombinant Ad5 GLP1 adenovirus significantly inhibited the pro-tumor inflammation mediated by inflammatory factors. In human and mouse liver cancer cells, Ad5 GLP1-infected tumor cells had a significant inhibitory effect on the activation of inflammatory pathways such as IL-6-induced TNF-α / NFkB and IL-6 / STAT3.

[0115] See also Fig. 9 The results showed that GLP1 protein significantly promoted anti-tumor immunity induced by oncolytic adenovirus. In the mouse liver cancer (Hepa1-6) model, the number of lymphocytes secreting IFN-γ was significantly increased in the GLP1 combined with oncolytic adenovirus treatment group.

[0116] See also Fig.10 The results showed that the recombinant Ad5 GLP1 adenovirus significantly inhibited the growth of liver cancer without obvious toxic side effects. After AD5 GLP1 treatment of mice bearing human liver cancer cells (LM3), tumor growth was significantly inhibited, with significant differences compared with mice in the control virus treatment group.

[0117] See also Fig.11 The results showed that the recombinant Ad5 GLP1 adenovirus significantly prolonged the survival time of liver cancer-bearing mice. After human liver cancer cell-bearing mice were treated with Ad5 GLP1, the survival time of the mice was significantly longer than that of the non-treated group and the control virus-treated group.

[0118] See also Fig.12 The results showed that the recombinant Ad5 GLP1 adenovirus significantly inhibited the growth of liver cancer without obvious toxic side effects. After Ad5 GLP1 treatment of mice bearing mouse liver cancer cells (H22), tumor growth was significantly inhibited compared with the control group mice.

[0119] See also Fig.13 The results showed that the recombinant Ad5 GLP1 adenovirus significantly inhibited the growth of pancreatic cancer without obvious toxic side effects. Compared with the control group, the tumor growth of mice bearing pancreatic cancer cells (Panc02) was significantly inhibited after Ad5 GLP1 treatment, and about 33% of the mice were cured.

[0120] See also Fig.14 The results showed that the recombinant Ad5 GLP1 adenovirus significantly prolonged the survival time of pancreatic cancer-bearing mice. After Ad5 GLP1 treatment, the survival time of pancreatic cancer-bearing mice was significantly longer than that of the control group, and about 40% of the mice survived for a long time.

[0121] From the above results, it can be seen that the present invention provides a replicative oncolytic adenovirus AD5 GLP1 that can inhibit tumor progression and significantly prolong the survival time of tumor-bearing individuals. The virus has a stronger replication ability in tumor cells than the wild-type virus. At the same time, the virus can highly express GLP1, which can be secreted outside the cell to exert its biological function.

[0122] The replicating oncolytic adenovirus Ad5 GLP1 of the present invention has a significant ability to inhibit tumor growth and prolong survival, and has a significant anti-tumor effect. One virus integrates multiple unique anti-tumor mechanisms in one, including regulating metabolism, promoting anti-tumor immunity, inhibiting tumor-promoting inflammation, etc., and has unexpected anti-tumor effects. It can be used to prepare anti-tumor drugs.

[0123] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

[0124] DNA sequence of GLP1-E1A (SEQ ID NO: 1)

[0125] GAC GGATCCGCCACCATGAAAAGCATTTACTTTGTGGCTGGATTATTTG TAATGCTGGTACAAGGCAGCTGGCAACATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCT GGTGAAAGGCCGAGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGG CTGGAGACGTGGAGGAGAACCCTGGACCTATGAGACATATTATCTGCCACGGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCG AAGAGGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGACGTGACGGCCCCCGAAGATCCCAACGA GGAGGCGGTTTCGCAGATTTTTCCCGACTCTGTAATGTTGGCGGTGCAGGAAGGGATTGACTTACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCC TCACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTTGGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCTTACCTGCCACGAGGC TGGCTTTCCACCCAGTGACGACGAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGGAGCACCCCGGGCACGGTTGCAGGTCTTGTCATTATCACCG GAGGAATACGGGGGACCCAGATATTATGTGTTCGCTTTGCTATATGAGGACCTGTGGCATGTTTGTCTACAGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAG CCAGAACCGGAGCCTGCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGCCCGACATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGAGTTGGTGGGCGTCGC CAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGCCTGGGCAACCTTTGGACTTGAGCTGTAAACGCCCCAGGCCATAAAGATCTCACCTATCGATAAGCTTGGGAGTTCCGGTCGACCTCGAGGGGCCC ctcgag CcC

[0126] SEQ ID NO:2

[0127] HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR. Sequence Listing <110> Nanjing Weiyade Biopharmaceutical Co., Ltd. <120> A replicating oncolytic adenovirus capable of inhibiting tumor progression and prolonging the survival time of tumor-bearing individuals and its application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1161 <212> DNA <213> Artificial Sequence <400> 1 gacggatccg ccaccatgaa aagcatttac tttgtggctg gattatttgt aatgctggta 60 caaggcagct ggcaacatgc tgaagggacc tttaccagtg atgtaagttc ttatttggaa 120 ggccaagctg ccaaggaatt cattgcttgg ctggtgaaag gccgaggaag cggagctact 180 aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc tatgagacat 240 attatctgcc acggaggtgt tattaccgaa gaaatggccg ccagtctttt ggaccagctg 300 atcgaagagg tactggctga taatcttcca cctcctagcc attttgaacc acctaccctt 360 cacgaactgt atgatttaga cgtgacggcc cccgaagatc ccaacgagga ggcggtttcg 420 cagatttttc ccgactctgt aatgttggcg gtgcaggaag ggattgactt actcactttt 480 ccgccggcgc ccggttctcc ggagccgcct cacctttccc ggcagcccga gcagccggag 540 cagagagcct tgggtccggt ttctatgcca aaccttgtac cggaggtgat cgatcttacc 600 tgccacgagg ctggctttcc acccagtgac gacgaggatg aagagggtga ggagtttgtg 660 ttagattatg tggagcaccc cgggcacggt tgcaggtctt gtcattatca ccggaggaat 720 acgggggacc cagatattat gtgttcgctt tgctatatga ggacctgtgg catgtttgtc 780 tacagtcctg tgtctgaacc tgagcctgag cccgagccag aaccggagcc tgcaagacct 840 acccgccgtc ctaaaatggc gcctgctatc ctgagacgcc cgacatcacc tgtgtctaga 900 gaatgcaata gtagtacgga tagctgtgac tccggtcctt ctaacacacc tcctgagata 960 cacccggtgg tcccgctgtg ccccattaaa ccagttgccg tgagagttgg tgggcgtcgc 1020 caggctgtgg aatgtatcga ggacttgctt aacgagcctg ggcaaccttt ggacttgagc 1080 tgtaaacgcc ccaggccata aagatctcac ctatcgataa gcttgggagt tccggtcgac 1140 ctcgaggggc ccctcgagcc c 1161 <210> 2 <211> 30 <212> PRT <213> Artificial Sequence <400> 2 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30

Claims

1. A replicating oncolytic adenovirus capable of inhibiting tumor progression and prolonging the survival of tumor-bearing individuals, characterized in that: The oncolytic adenovirus comprises a nucleotide sequence encoding a glucagon-like peptide-1 receptor agonist; the oncolytic adenovirus can selectively replicate and dissolve tumors in tumors, and can also replicate and express a glucagon-like peptide-1 receptor agonist protein, which can regulate tumor microenvironment metabolism and inhibit tumor-promoting inflammation; the amino acid sequence of the glucagon-like peptide-1 receptor agonist is the amino acid sequence shown in SEQ ID NO: 2, and the oncolytic adenovirus is a type 5 adenovirus.

2. The replicative oncolytic adenovirus according to claim 1, characterized in that The glucagon-like peptide-1 receptor agonist is a secretory protein glucagon-like peptide-1, which can promote the replication of oncolytic adenovirus.

3. Use of the replicative oncolytic adenovirus according to any one of claims 1 to 2 in the preparation of anti-tumor drugs.

4. The use according to claim 3, characterized in that: The tumor is one of liver cancer, pancreatic cancer and colon cancer.

5. A pharmaceutical composition comprising the replicative oncolytic adenovirus according to any one of claims 1-2, and a pharmaceutically acceptable excipient.

6. The method for constructing a replicative oncolytic adenovirus for inhibiting tumor progression and prolonging the survival time of tumor-bearing individuals according to any one of claims 1-2, characterized in that: This is achieved through three steps: plasmid construction, virus rescue and virus amplification.

7. The method for constructing a replicative oncolytic adenovirus that inhibits tumor progression and prolongs the survival time of tumor-bearing individuals according to claim 6, characterized in that: The specific steps include: Step 1, plasmid construction: linearize the constructed shuttle vector pShuttle-GLP1 with PmeI and transfer it into competent medium pAdEasy-BJ5183, screen positive clones for culture and identification, and re-transform the correct clone plasmid into DH5a competent medium for secondary screening and identification. After the correct identification, perform large-scale plasmid extraction to obtain the full-length plasmid of Ad5 GLP1; Step 2, virus rescue: The full-length plasmid of Ad5 GLP1 was linearized with PacI, purified and transfected into 293T cells until 80% of the cells showed pathological changes. The cells were blown off with culture medium and collected into centrifuge tubes. After repeated freeze-thaw cycles, the supernatant of the virus was collected and stored at -80°C as virus seed; Step 3, virus amplification: Take the virus seed solution and add it to the 293T cell plate for culture. When the cell density reaches above 90%, subculture at a ratio of 1 to 3 until 80% of the cells show pathological changes. Collect the virus according to the method in step 2 and purify the virus by centrifugation.

8. The method according to claim 7, characterized in that The construction method of the shuttle vector pShuttle-GLP1 is as follows: first, the DNA sequence of GLP1-E1A is synthesized, as shown in SEQ ID NO: 1; the aforementioned sequence is double-digested with BamHI and XhoI, and the pShuttle plasmid is also double-digested with BamHI and XhoI; the DNA after digestion and the pShuttle plasmid are connected with T4 ligase; the connection product is transformed into DH5a competent state, and positive clones are screened on kanamycin-resistant LB plates; positive monoclonal DH5a bacteria are amplified, and plasmids are extracted; this plasmid is the shuttle-GLP1 plasmid used to prepare pAd5 GLP1 adenovirus.

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