WO2022170670A1

WO2022170670A1 - Replicative oncolytic adenovirus capable of inhibiting tumor progression and prolonging survival time of tumor-bearing individual, and use thereof

Info

Publication number: WO2022170670A1
Application number: PCT/CN2021/083176
Authority: WO
Inventors: 魏继武; 董杰; 吴静怡; 陆贤
Original assignee: 南京惟亚德生物医药有限公司
Priority date: 2021-02-09
Filing date: 2021-03-26
Publication date: 2022-08-18
Also published as: CN114908064A

Abstract

Provided are a replicative oncolytic adenovirus Ad5GLP1 capable of inhibiting tumor progression and prolonging the survival time of a tumor-bearing individual, and the use thereof. Provided is a method for designing and constructing Ad5GLP1. The replicative oncolytic adenovirus Ad5GLP1 is successfully obtained, and the virus can be specifically replicated in tumor cells and secreted out of the cells, the glucagon-like peptide-1 receptor agonist-glucagon-like peptide 1 (GLP 1) can be highly expressed, and a large amount of protein molecules can be secreted out of the cell and virus replication can be significantly promoted. The new replicative oncolytic adenovirus has the effects of significantly enhancing anti-tumor immunity, inhibiting solid tumor growth and prolonging the survival of tumor-bearing individuals, and has great prospects and value for developing anti-tumor drugs.

Description

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

technical field

The present invention relates to the field of tumor biological therapy, in particular to a replicative oncolytic adenovirus capable of inhibiting tumor progression and prolonging the survival time of tumor-bearing individuals and its application.

Background technique

Cancer has become the number one disease endangering human life and health. In my country, about 4 million new cancer patients are added every year, and nearly 3 million people die of cancer every year. Surgery is the most effective treatment for early-stage cancer, and it does not significantly improve the vast majority of patients with advanced and metastatic disease. Although radiotherapy/chemotherapy and other adjuvant therapy can inhibit tumor progression to a certain extent, the overall survival rate of cancer patients has not been significantly improved. Searching for new, safe and effective therapeutic drugs is the focus of pharmaceutical research all over the world today.

In recent years, tumor immunotherapy has attracted much attention due to its remarkable curative effect. Tumor immunotherapy is a therapeutic approach that activates the body's immune system to kill and monitor cancer cells by activating and/or regulating immune-related pathways. As a new star in the field of tumor immunotherapy, oncolytic virus T-Vec was approved by the FDA at the end of 2015, and oncolytic virus-mediated anti-tumor immunotherapy has received more and more attention. Oncolytic virus is a kind of virus with self-replication ability and a large number of replication in tumor cells. It has a variety of unique anti-tumor effects: it can induce the immunogenic death of tumor cells; activate the immune surveillance of the body; express recombinant proteins to regulate tumors Microenvironment. At present, clinical trials related to a variety of oncolytic viruses are being carried out, such as adenovirus, herpes simplex, measles virus, vaccinia virus, herpes stomatitis virus, etc., and good therapeutic effects have been achieved.

However, solid tumors escape the immune surveillance of the body and promote invasion and metastasis through a variety of mechanisms, including abnormal metabolism and a pro-inflammatory microenvironment. Glucagon-like peptide 1 (GLP1) is a polypeptide consisting of 30 amino acid residues. It is a glucagon-like peptide-1 receptor agonist, which can not only reduce blood sugar, but also significantly inhibit multiple tumor-promoting inflammations. The role of the pathway, including the inhibition of the activation of NF-κB/IL-6/STAT3 pathway, the production of NLRP3/IL-1β inflammasome, etc.

Studies have found that while oncolytic virus activates immunity, it also upregulates tumor-promoting inflammation in the tumor microenvironment and changes the metabolic microenvironment, which is not conducive to its anti-tumor effect. How to effectively overcome the inherent deficiencies in oncolytic virus therapy is a Important scientific problems to be solved by the present invention.

SUMMARY OF THE INVENTION

In view of the deficiencies of 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 It is secreted outside the cell, and the glucagon-like peptide-1 receptor agonist expressed by the virus can be secreted outside the cell and promote virus replication, inhibit tumor-promoting inflammation, and significantly inhibit tumor growth; finally, the invention establishes a variety of Tumor models, including liver cancer, pancreatic cancer, etc., verify the significant effect of oncolytic adenovirus therapy on tumor prolongation.

The technical scheme adopted by the present invention to solve the technical problem is:

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

Among them, the oncolytic adenovirus can selectively replicate and oncolyze in tumors, and can replicate and express a glucagon-like peptide-1 receptor agonist, which can regulate tumor microenvironment metabolism and inhibit tumor-promoting inflammation.

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

(1) amino acid sequence as described in SEQ ID NO: 2;

(2) An amino acid sequence that has more than 80% homology with the sequence described in SEQ ID NO: 2 and is related to oncolysis.

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

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

The present invention also protects the application of the above-mentioned replicative oncolytic adenovirus in the preparation of antitumor drugs.

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

The present invention also protects a pharmaceutical composition comprising the replication-type oncolytic adenovirus described above, and a pharmaceutically acceptable excipient.

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 of plasmid construction, virus rescue and virus amplification.

Preferably, the following specific steps are included:

Step 1. Plasmid construction: The constructed shuttle vector pShuttle-GLP1 was linearized with PmeI and then transformed into competent pAdEasy-BJ5183, screened for positive clones, cultured and identified, and the correct cloned plasmid was identified and transformed into DH5a competent for secondary screening , after the identification is correct, the plasmid is extracted to obtain the Ad5GLP1 full-length plasmid;

Step 2, virus rescue: Ad5 GLP1 full-length plasmid was linearized with PacI, purified and transfected into 293T cells. When 80% of the cells became diseased, the cells were blown down with medium and collected into a centrifuge tube. After repeated freezing and thawing, the cells were centrifuged to collect the virus. The supernatant was stored at -80°C as a 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 more than 90%, pass 1 to 3 passages until 80% of the cells become diseased, collect the virus according to the method of step 2, and purify by centrifugation. Virus.

More preferably, the construct 6CD5 of the shuttle vector Ad5-pShuttle-GLP1 is as follows: first, the DNA sequence of GLP1-E1A is synthesized by gene, as shown in SEQ ID NO: 1; the aforementioned sequence is double digested by BamHI and XhoI, while pShuttle The plasmid was also double digested with BamHI and XhoI; the digested DNA and pShuttle plasmid were ligated with T4 ligase; the ligated product was transformed into DH5a competent, and positive clones were screened on kanamycin-resistant LB plates; amplification The positive monoclonal DH5a bacteria were extracted, and the plasmid was extracted; this plasmid was the shuttle-GLP1 plasmid used to prepare the pAd5 GLP1 adenovirus.

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

The invention also protects the application of the soluble protein GLP1 in the preparation of oncolytic adenovirus replication-promoting and oncolytic drugs.

The invention also protects the application of the soluble protein GLP1 in the preparation of antitumor drugs.

The invention also protects the application of the soluble protein GLP1 in the preparation of drugs for inhibiting the inflammatory pathway.

beneficial effect

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

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

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

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

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

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

(6) Ad5 GLP1 can significantly promote the antitumor immune response induced by oncolytic adenovirus.

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

(8) In animal experiments, Ad5 GLP1 significantly inhibited the growth of pancreatic cancer and significantly prolonged the survival time of mice.

Description of drawings

Figure 1 shows the construction strategy of the replication-type oncolytic adenovirus Ad5 GLP1 according to the results of the present invention; wherein, A is the schematic diagram of the construction method of Ad5 GLP1 virus; B is the PCR result of the target gene GLP1, wherein 1 is the PCR product, and 2 is the DL2000 DNA Maker.

In Figure 2, (A) LM3 human hepatoma cells and (B) Hepa1-6 mouse hepatoma 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 harvested, and the expression of the target protein GLP1 was detected by WB. Data are representative of three independent replicates. GFP expression was able to show viral replication.

Figure 3 is the effect of the soluble protein GLP1 of the present invention on the replication of oncolytic adenovirus, A is 48 hours, B is 72 hours. Hepatoma LM3 cells were inoculated into 96-well culture plates, treated with adenovirus expressing luciferase (MOI=5) with or without GLP1 (20 μM), and then continued to culture, and 48 and 72 hours later, respectively, were added to detect the luciferase substrate. and detect the fluorescence intensity.

Figure 4 is a comparison of the replication ability of Ad5 GLP1 of the present invention in tumor cells and the control virus Ad5 con. Colon cancer MC38 cells were infected with Ad5 con and Ad5 GLP1 at MOI of 1, respectively. The cells were harvested at 12, 24, 48, 60 and 72 hours, and total RNA was extracted. Quantitative RT-PCR was used to detect the gene expression of the virus-specific protein Hexon. Level. The data are expressed as a 12-hour expression value of 1, respectively, showing the fold increase in viral replication.

Figure 5 shows the effect of the secreted protein GLP1 of the present invention on the oncolytic effect of oncolytic adenovirus; wherein, A is the effect on cell viability, and B is the effect on the number of viable cells. The HCC-LM3 hepatoma cells were seeded in 96-well or 6-well plates, and after they adhered, in the presence or absence of GLP1 (20 μM), the hepatoma cells HCC-LM3 were infected with adenovirus with an MOI value of 5. , untreated group and single GLP1-treated group were used as controls. After culturing for 48 h, the proportion of viable cells was detected by MTT method or the number of viable cells after trypan blue staining was detected by 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 .

Figure 6 shows that the soluble GLP1 of the present invention significantly inhibits the activation of inflammatory pathways induced by inflammatory factors or oncolytic adenovirus. Biological function verification of anti-inflammatory peptide genetic engineering plasmid GLP1 A. Human or mouse hepatoma cells were inoculated in 6-well plates, and after adherence, transfected with GLP1 plasmid or blank plasmid respectively, and 24h later, IL-6 (25ng/mL) was added. ml) after stimulation for 0.5 h, the cell protein was collected, and the phosphorylation level of STAT3 was detected by Western Blot method. B. Human hepatocellular carcinoma cells HCC-LM3 were inoculated in 6-well plates. After adhering to the wall, they were transfected with GLP1 plasmid or blank plasmid respectively. After 24 hours, adenovirus (MOI=5) was added. After co-cultivation for 24 hours, cell RNA was extracted. Reverse transcription was made into cDNA, and 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 .

Figure 7 shows that the recombinant oncolytic adenovirus Ad5 GLP1 of the present invention significantly inhibits the activation of the virus-induced pro-tumor inflammatory pathway, wherein A corresponds to HCC-LM3 cells, and B corresponds to Hepa1-6 cells. Hepatocellular carcinoma cells HCC-LM3 and Hepa1-6 cells were inoculated in 6-well plates, respectively, and the cells were infected with recombinant oncolytic adenovirus (MOI=5) for 24 h after adherence. The cells were harvested and the protein and RNA were extracted respectively. and qRT-PCR to detect the levels of corresponding inflammatory factors. 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 .

Figure 8 shows that 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 seeded respectively. After 24 hours, IL-6 (25ng/ml) was added to the medium for stimulation for 0.5 hours, and the cell proteins were collected. Western blot was used to detect the expression of STAT3 and IκBα. phosphorylation level.

Figure 9 shows that the secreted protein GLP1 of the present invention enhances the anti-tumor immune response of recombinant oncolytic adenovirus. The mouse liver cancer Hepa1-6 cells were used to construct a mouse subcutaneous tumor model. When the mouse tumor grew to 0.4 cm × 0.4 cm, the mice were randomly divided into 4 groups, and the treatment group was given GLP1 (300 μg/kg/ Only) intratumoral injection, adenovirus Ad5 treatment group was given adenovirus 5×10 ⁸ PFU per mouse, intratumoral injection, once a day, combined treatment group was given GLP1 and adenovirus treatment at the same time, control group was given the same volume of PBS tumor One week later, the mice were sacrificed by cervical dislocation, the tumor tissue was isolated, minced and digested with collagenase, then sieved into a single cell suspension, and the amount and intensity of IFN-γ released by immune cells were detected by ELISpot method.

Figure 10 shows that 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 protocol. Mice were inoculated with 1 × 10 ⁷ / HCC-LM3 cells 4 days before treatment (-4 days), the tumor size and body weight of mice were measured for the first time from day 0, and the mice were randomly divided into 3 groups, including Control group, Ad5 GLP1 group and Ad5 con group, 6-7 animals in each group. The treatment group was injected with Ad5 GLP1, 1×10 ⁹ PFU/mice, or the same dose of Ad5 con, the control group was injected with the same volume of PBS, and adenovirus was injected every 5 days until the tumor volume of the mice was greater than 2.0 cm ³ or mice died. B. The injection of adenovirus was 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 injections were started on day 0, and mice were weighed every two days. The figure shows the body weight of mice in each group. The results are mean (Mean) + standard deviation (SD), #P>0.05;*P<0.05;**P<0.01.

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

Figure 12 shows that the recombinant oncolytic adenovirus Ad5 GLP1 of the present invention can significantly inhibit the growth of liver cancer in immune-competent mice without obvious side effects. Balbc male mice were inoculated with 5x10 ⁶ H22 hepatoma cells, and after the tumor grew to about 5x7mm in size, 3x10 ⁸ pfu Ad5 GLP1 was injected intratumorally, once every other day for three times, and the tumor diameter (left) and the change in mouse body weight ( right).

Figure 13 shows that the recombinant oncolytic adenovirus Ad5 GLP1 of the present invention can significantly inhibit the growth of pancreatic cancer in immune-competent mice without obvious negative effects. Balbc male mice were inoculated with 5x10 ⁶ Panc02 pancreatic cancer cells. After the tumor grew to about 5x7mm in size, 3x10 ⁸ pfu Ad5 GLP1 was injected intratumorally, once every other day for a total of three times, and the tumor diameter (left) and the body weight of the mice were measured every other day. (right).

Figure 14 shows that the recombinant oncolytic adenovirus Ad5 GLP1 of the present invention can significantly prolong the survival time of pancreatic cancer tumor-bearing mice, and 33% of the mice were cured and survived for a long time. Balbc male mice were inoculated with 5x10 ⁶ Panc02 pancreatic cancer cells, and after the tumor grew to about 5x7 mm in size, 3x10 ⁸ pfu Ad5 GLP1 was injected intratumorally, once every other day for a total of three times, and the survival time of the mice was monitored.

Detailed ways

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

1 Experimental materials and methods

1.1 Experimental materials and instruments

1.1.1 Experimental cell lines

Human embryonic kidney cell line 293T, human liver cancer cell line HCC-LM3, mouse liver cancer cell line Hepa1-6 and H22, mouse pancreatic cancer cell line Panc02, mouse colon cancer cell line MC38, using 10% fetal bovine serum, High glucose DMEM medium with 100 U/I penicillin and 1 mg/ml streptomycin was cultured in a 37°C, 5% _CO2 incubator.

1.1.2 Experimental Instruments

biological safety cabinet (

advance, Class II Biological Safety Cabinet, The Baker Company), carbon dioxide incubator (FORMA SERIES II WATER JACKET CO ₂ incubator, Thermo), cryogenic centrifuge (HERAEUS MEGAFUGE 1.0R, Thermo), vertical electrophoresis tank (BIO-RAD), Electrophoresis instrument (BIO-RAD), semi-dry transfer-film transfer instrument (BIO-RAD), Western blot exposure system (Alpha Innotech), PCR instrument (PCR Thermal Cycler Dice, TaKaRa), real-time quantitative PCR instrument and analysis software (ABI384) , Sequence Detection Software, Version 1.3.1), microplate reader (VERSA max microplate reader), complete set of pipettes (eppendorf and RAININ), cell counter (Countstar Automated cell counter, Inno-Alliance Biotech Inc., Wilmington, USA ), flow cytometer (FACSCalibur, Becton, Dickinson and Company, USA), FlowJo software (Version 7.6.5, Tree Star Inc, Ashland, Oregon), microplate shaker (QiLinBeiEr), nucleic acid purity concentration detector ( Biophotometer plus, eppendorf), digital constant temperature water bath (Guohua Electric).

1.1.3 Main experimental reagents and consumables

Primers were synthesized by GenScript. The DMEM high glucose medium, double antibody and serum required for tumor cell culture were purchased from Invitrogen (Shanghai) Company. Quantitative RT-PCR reagent Faststart Universal SYBR Green Master (Roche, 04913914001). Reagents and consumables required for Western Blot: protease inhibitor (Roche, 11873580001), cell lysate (Biyuntian: P0013), PVDF membrane (Roche, 03010040001), WB Immobilon ECL luminescent solution (Millipore, WBKLS0500), primary antibody dilution ( Biyuntian, P0023A), HRP-labeled secondary antibody (Multisciences, GAR007 and GAM007, 1:5000 dilution), and other required reagents were domestic analytical grades, purchased from the School of Chemistry and Chemical Engineering, 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). .

1.2 Experimental method

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

A. Ad5 GLP1 full-length plasmid construction:

First, the DNA sequence of GLP1-E1A was genetically synthesized, as shown in SEQ ID NO: 1; the aforementioned sequence was double digested with BamHI and XhoI, and the pShuttle plasmid was also double digested with BamHI and XhoI; the digested DNA and pShuttle plasmid T4 ligase was used for ligation; the ligation product was transformed into DH5a competent, and positive clones were screened on kanamycin-resistant LB plates; positive monoclonal DH5a bacteria were amplified, and plasmid extraction was performed. This plasmid is the shuttle-GLP1 plasmid used to make pAd5 GLP1 adenovirus.

The constructed plasmid pShuttle-GLP1 was linearized with PmeI and then transformed into competent pAdEasy-BJ5183. The LB plate containing 50ug/ml kanamycin was used for screening, and the positive clones were picked and cultured for identification. The DH5a competent cells were transformed for secondary screening and identification. After the identification was correct, plasmid extraction was carried out to obtain the Ad5 GLP1 full-length plasmid.

B. Ad5 GLP1 virus rescue:

Ad5 GLP1 full-length plasmid was linearized with PacI. After purification, 293T cells were transfected into 1 ug/well in 6-well plates, and cultured at 37°C with 5% CO ₂ . After 2 days, the cells were digested and transferred to 10 cm dishes, and the medium was changed for 2-3 days. , until 80% of cells become diseased, use 10ml of medium to blow down the cells and collect them into a 15ml centrifuge tube, freeze and thaw twice, centrifuge at 3000rpm/min for 15min, collect the virus supernatant and store it at -80°C as a virus seed.

C. Viral Amplification:

Take 50ul of virus seed solution and add 60% 293T cells to a 10cm dish, incubate at 37°C with 5% CO ₂ , the cell density reaches more than 90%, and pass 1 to 3 passages until 80% of the cells become diseased, and there are about 10 cells in the dish. The virus was collected according to the above method, and purified by cesium chloride density gradient centrifugation; the titer was determined by the TCID50 method.

Evaluation of Ad5 GLP1 Virus Function

A. Expression and secretion function of GLP1:

72 hours after Ad5 GLP1 virus infection of tumor cells, the cells and supernatant were harvested, and the expression and secretion function of GLP1 were detected by WB.

B. Virus replication ability:

Ad5 GLP1 and Ad5 con (Ad5 con is a type 5 adenovirus that only contains the E1A sequence of the viral replication original and does not contain the GLP1 sequence) virus infected tumor cells at the same MOI. The cells were harvested at different times, and the same amount of virus suspension was obtained after repeated freezing and thawing centrifugation. 293T cells were used for virus titer determination; changes in virus replication ability were analyzed.

C. Oncolytic function:

Tumor cells were infected with Ad5 GLP1 and Ad5 con viruses at an MOI of 1 to 100, respectively, and the cell viability was detected by MTT 72 hours later to evaluate the tumoricidal effect of Ad5 GLP1.

1.2.2 In vivo study of the anti-tumor effect and mechanism of Ad5 GLP1

A. Select 6-8 week old Balb/c or C57BL/6 mice to establish a subcutaneous tumor model in the right armpit or subcutaneously. After 4-6 days, the tumor size was measured to 200mm ³ , and the mice were randomly divided into 3 groups, namely: No treatment group, control Ad5 con virus treatment group, Ad5 GLP1 virus treatment group; intratumoral injection of the corresponding virus was used according to the group, and the amount of virus injected per mouse was 2.5×10 ⁸ pfu, and the tumor volume and body weight were tracked and measured. After the mice died naturally, The mouse survival time was recorded.

1.2.3 Titer determination of Ad5 GLP1 virus

1) 293T cells were seeded in a 96-well plate, about 1×10 ³ cells per well, and the titer was determined after the cells adhered.

2) Dilution of virus gradient: 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; Pipette 50 μl from the tube into the second EP tube, mix well, mark it as 10-2; and so on, until the dilution reaches the desired gradient.

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

4) After 5 days, the 96-well plate was placed under a microscope to observe GFP, and the number of wells with GFP in each gradient was recorded for the calculation of virus titer.

5) Calculation formula of virus titer TCID50:

Log10(TCID50)=L+d(s- _0.5 )+log10(1/v);

Wherein, L=Log10 highest dilution (if the highest dilution is 10 times dilution, L=1);

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

d=Log10 dilution (if it is 10-fold dilution, d=1);

s=sum of individual gradient GFP ratios.

1.2.4 Quantitative PCR

The 10 μl system of real-time quantitative PCR consists of 2.6 μl PCR water, 0.2 μl upstream and downstream primers, 2 μl template and 5 μl SYBR Green fluorescent dye. The samples were mixed and amplified on an ABI 384 PCR machine.

1.2.5 Extraction and concentration determination of total cell protein

1) Take 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 pipet the cells, put the cells into an EP tube, and centrifuge at 1500 rpm for 5 min.

2) Remove the supernatant, add PBS to resuspend the cells, and centrifuge at 1500 rpm for 5 min.

3) Remove PBS, add the corresponding cell lysate containing protease inhibitor to each well according to the amount of cells, vortex for 30s, place on ice for 10min, and repeat the operation three times. Centrifuge at 12000g for 15min at 4°C. Collect the supernatant in another clean EP tube.

4) Determination of protein concentration: Detect according to the instructions of the BCA protein concentration assay kit. Take 2μl of protein sample in 96-well plate, add 18μl of PBS to dilute the sample, and finally add 200μl of assay working solution (working solution is composed of reagent A:reagent B=50:1), placed in an oven at 60°C, after 30min , measure the absorbance at 562nm with a microplate reader, and calculate the concentration of the protein sample according to the standard curve.

5) Add 1/4 of the volume of protein lysate to each tube of 5× loading buffer, mix well, and place in a metal bath at 100°C for 5 minutes. After cooling, store at -20°C for later use.

1.2.6 Western blot experiment

1) Gel preparation and electrophoresis: SDS-PAGE separating gel and stacking gel with different concentrations were prepared according to different requirements. According to the calculation result of protein quantification, the loading amount of each sample was adjusted to 30 μg. Electrophoresis conditions: stacking gel at 80V for 30min, separating gel at 120V for about 80min, provided that the bands are separated and will not run out.

2) Transfer membrane: prepare filter paper and PVDF membrane, first soak PVDF membrane with methanol, and then soak in membrane transfer buffer together with filter paper for later use. Carefully remove the glue from the glass plate, soak it in transfer buffer, and place it in the sandwich order of negative electrode-filter paper-PVDF membrane-glue-filter paper-positive electrode to drive away air bubbles. 110mA transfer membrane for 60-70min.

3) Blocking: Immediately after membrane transfer, the PVDF membrane was taken out and placed in 5% nonfat milk powder for blocking at room temperature for 1 hour.

4) Primary antibody incubation: Incubate the primary antibody overnight at 4°C.

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

6) Exposure: Wash the strips with washing buffer, 10 min each time, three times in total; expose the strips with chemiluminescence solution on a WB exposure apparatus, and obtain strip images.

1.2.7 Trypan blue count

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

2. Results and Conclusions

Referring to Figure 2, the results show that the replicative oncolytic adenovirus Ad5 GLP1 constructed by us has the same ability to infect tumor cells and express the target gene compared with the control virus. In human and mouse liver cancer cell lines HCC-LM3 and Hepa1-6 cell lines, Ad5 GLP1 can efficiently infect cells and express GLP1 protein.

Referring to Figure 3, the results show that soluble GLP1 can significantly promote the replication of oncolytic adenovirus in tumor cells. In the medium containing GLP1 protein, the replication of oncolytic adenovirus (Ad5) in hepatoma cells (LM3) was significantly increased, indicating that GLP1 has the effect of promoting the replication of oncolytic adenovirus.

Referring to Figure 4, the results show that the replication of recombinant oncolytic adenovirus Ad5 GLP1 in tumor cells is significantly higher than that of control oncolytic adenovirus. In mouse colon cancer cells, the mouse colon cancer cell line (MC38) was infected with Ad5 GLP1 at MOI of 1, and the gene expression number of Hexon of Ad5 GLP1 was significantly higher than 48, 60 and 72 hours after infection of cells. The copy number of the control virus indicated that the replication ability of Ad5 GLP1 in the tumor was significantly enhanced.

Referring to Figure 5, the results show that soluble GLP1 can promote oncolytic adenovirus oncolysis. In the medium containing GLP1 protein, the oncolytic effect of adenovirus on hepatoma cells (LM3) was significantly higher than that of the virus or GLP1 alone treatment group, indicating that GLP1 promotes adenovirus oncolysis.

Referring to Figure 6, the results show 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).

Referring to Figure 7, the results show that recombinant Ad5 GLP1 adenovirus can significantly inhibit virus-induced activation of pro-tumor inflammatory pathways. In human and mouse hepatoma cell lines, the TNF-α/NFkB and IL-6/STAT3 pathways were significantly inhibited in the Ad5 GLP1 treatment group.

Referring to Figure 8, the results show that recombinant Ad5 GLP1 adenovirus significantly inhibited pro-tumor inflammation mediated by inflammatory factors. In human and mouse hepatoma cells, Ad5 GLP1-infected tumor cells significantly inhibited IL-6-induced activation of inflammatory pathways such as TNF-α/NFkB and IL-6/STAT3.

Referring to Figure 9, the results show that GLP1 protein significantly promotes oncolytic adenovirus-induced anti-tumor immunity. In the mouse liver cancer (Hepa1-6) model, GLP1 combined with oncolytic adenovirus treatment group significantly increased the number of IFN-γ-secreting lymphocytes.

Referring to Figure 10, the results show that the recombinant Ad5 GLP1 adenovirus significantly inhibited the growth of liver cancer without obvious toxic side effects. After AD5 GLP1 treatment of human hepatoma cell (LM3) tumor-bearing mice, tumor growth was significantly inhibited, which was significantly different from that of control virus-treated mice.

Referring to Figure 11, the results show that recombinant Ad5 GLP1 adenovirus significantly prolongs the survival time of liver cancer tumor-bearing mice. After the human hepatoma cell tumor-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.

Referring to Figure 12, the results show that the recombinant Ad5 GLP1 adenovirus significantly inhibited the growth of liver cancer without obvious toxic side effects. Mouse hepatoma cell (H22) tumor-bearing mice treated with Ad5 GLP1 significantly inhibited tumor growth compared with control mice.

Referring to Figure 13, the results show that the recombinant Ad5 GLP1 adenovirus significantly inhibited the growth of pancreatic cancer without obvious toxic side effects. In mouse pancreatic cancer cell (Panc02) tumor-bearing mice treated with Ad5 GLP1, tumor growth was significantly inhibited compared with control mice, and about 33% of mouse tumors were cured.

Referring to Figure 14, the results show that recombinant Ad5 GLP1 adenovirus significantly prolongs the survival time of pancreatic cancer tumor-bearing mice. After treatment with Ad5 GLP1 in pancreatic cancer-bearing mice, the survival time of mice was significantly longer than that of control mice, and about 40% of the mice survived for a long time.

From the above results, 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 ability to replicate in tumor cells than the wild-type virus. At the same time, the virus can highly express GLP1, and the protein can be secreted outside the cell to exert its biological function.

The replication-type oncolytic adenovirus Ad5 GLP1 of the present invention has a significant ability to inhibit tumor growth, prolong the survival period, and has a significant anti-tumor effect. A virus that integrates multiple unique anti-tumor mechanisms at the same time, including regulation of metabolism, promotion of anti-tumor immunity, inhibition of pro-tumor inflammation, etc., has unexpected anti-tumor effects. Can be used to prepare antitumor drugs.

The foregoing has shown and described 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 by the above-mentioned embodiments. The above-mentioned embodiments and descriptions only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Various changes and improvements, the claimed scope of the present invention is defined by the appended claims, description and their equivalents.

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

SEQ ID NO: 2

Claims

A replicative oncolytic adenovirus capable of inhibiting tumor progression and prolonging the survival time of tumor-bearing individuals, characterized in that the oncolytic adenovirus comprises a nucleotide sequence encoding a glucagon-like peptide-1 receptor agonist .
The replicative oncolytic adenovirus according to claim 1, wherein the oncolytic adenovirus can selectively replicate and oncolytic in tumors, and can replicate and express a glucagon-like peptide-1 receptor agonist at the same time A protein that regulates tumor microenvironment metabolism and inhibits tumor-promoting inflammation.
The replicative oncolytic adenovirus according to claim 1 or 2, wherein the amino acid sequence of the glucagon-like peptide-1 receptor agonist is selected from the following (1) or (2):

(1) amino acid sequence as described in SEQ ID NO: 2;

(2) An amino acid sequence that has more than 80% homology with the sequence described in SEQ ID NO: 2 and is related to oncolysis.
The replicative oncolytic adenovirus according to claim 1 or 2, wherein the glucagon-like peptide-1 receptor agonist is a secreted protein glucagon-like peptide 1, which can promote Oncolytic adenovirus replication.
Application of the replicative oncolytic adenovirus according to any one of claims 1 to 4 in the preparation of antitumor drugs.
The application according to claim 5, wherein the tumor is one of liver cancer, pancreatic cancer and colon cancer.
A pharmaceutical composition comprising the replicative oncolytic adenovirus according to any one of claims 1-4, and a pharmaceutically acceptable excipient.
The method for constructing a replicative oncolytic adenovirus capable of inhibiting tumor progression and prolonging the survival time of a tumor-bearing individual according to any one of claims 1-4, characterized in that it is achieved by three steps of plasmid construction, virus rescue and virus amplification .
The method for constructing a replicative oncolytic adenovirus capable of inhibiting tumor progression and prolonging the survival time of a tumor-bearing individual according to claim 8, wherein the method comprises the following specific steps:

Step 1, plasmid construction: linearize the constructed plasmid pShuttle-GLP1 with PmeI and then transform it into competent pAdEasy-BJ5183, screen for positive clones and culture identification, identify the correct cloned plasmid and re-transform DH5a competent for secondary screening and identification, After the identification is correct, the plasmid is extracted to obtain the Ad5 GLP1 full-length plasmid;

Step 2, virus rescue: Ad5 GLP1 full-length plasmid was linearized with PacI, purified and transfected into 293T cells. When 80% of the cells became diseased, the cells were blown down with medium and collected into a centrifuge tube. After repeated freezing and thawing, the virus was collected by centrifugation. The supernatant was stored at -80°C as a 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 more than 90%, pass 1 to 3 passages until 80% of the cells become diseased, collect the virus according to the method of step 2, and purify by centrifugation Virus.
The method according to claim 9, wherein the construction method of the shuttle vector Ad5-pShuttle-GLP1 is as follows: first, the DNA sequence of GLP1-E1A is synthesized by gene, as shown in SEQ ID NO: 1; Double digested with XhoI, and pShuttle plasmid was also digested with BamHI and XhoI; the digested DNA and pShuttle plasmid were ligated with T4 ligase; the ligation product was transformed into DH5a competent, and the kanamycin-resistant LB Screen the positive clones on the plate; amplify the positive monoclonal DH5a bacteria, and extract the plasmid; this plasmid is the shuttle-GLP1 plasmid used for the preparation of pAd5 GLP1 adenovirus.