Recombinant oncolytic adenovirus for expressing IL-36 as well as construction method and application of recombinant oncolytic adenovirus

Abstract

The invention discloses a recombinant oncolytic adenovirus for expressing IL-36 as well as a construction method and application thereof, and belongs to the technical field of biological medicines, and the recombinant oncolytic adenovirus H101 + IL-36 is constructed by integrating a human IL-36 gene into an oncolytic adenovirus H101 genome through a genetic engineering technology. The virus can specifically replicate and express IL-36 in tumor cells, synergistically activate congenital and adaptive immune response through a dual mechanism of oncolytic action and local immune regulation, effectively remodel a tumor immune microenvironment, and convert a cold tumor into a hot tumor. In-vitro and in-vivo experiments prove that H101 + IL-36 can significantly inhibit the proliferation of colon cancer cells, induce cell apoptosis, promote anti-tumor immunity and effectively inhibit the growth of mouse transplanted tumors. The recombinant oncolytic adenovirus constructed by the invention is applied to medicines for treating tumors such as hepatocellular carcinoma and the like, and has a wide clinical prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a recombinant oncolytic adenovirus expressing IL-36, a construction method thereof and application thereof in preparation of an anti-tumor drug. BACKGROUND

[0002] Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide, with approximately 1.9 million new cases annually, becoming a major public health problem. With changes in lifestyle (such as increased high-fat diet and reduced physical activity), its incidence continues to rise. CRC development involves a multi-stage evolution process, from normal epithelium to adenomatous polyps and finally to cancer, with significant genetic heterogeneity. Current treatment is mainly surgery combined with radiotherapy and chemotherapy, but the 5-year survival rate of patients with advanced disease is only 13.1%, and residual cancer cells after surgery often lead to recurrence.

[0003] Oncolytic viruses (OVs) are a new type of therapeutic approach that can selectively infect and lyse tumor cells, while triggering the body's anti-tumor immune response. Among them, adenovirus H101 (such as Ankorui ® ) has shown good safety and certain anti-tumor effect in clinical application. However, the response rate of single oncolytic virus therapy is still not ideal in some patients, and its efficacy is limited by the immunosuppressive state of the tumor microenvironment (TME), i.e. the so-called "cold" tumor.

[0004] Interleukin 36 (IL-36) is an important member of the IL-1 cytokine family, mainly including three agonists (IL-36α, IL-36β, IL-36γ) and one receptor antagonist (IL-36Ra). Its biological function is mediated by binding to the IL-36R receptor and recruiting the IL-1RAcP co-receptor, thereby activating the downstream NF-κB and MAPK signaling pathways, and playing a core role in inflammatory response and immune regulation. Studies have shown that IL-36 can directly act on dendritic cells (DCs), promoting their maturation and antigen-presenting ability, and can significantly enhance the activation, proliferation and cytotoxicity of CD8 + T cells, thereby remodeling the TME and converting "cold" tumors into "hot" tumors. These characteristics make IL-36 a highly potential immunotherapy candidate molecule.

[0005] However, when IL-36 is used as a systemic drug alone, there is a risk of triggering excessive inflammatory response, immune-related adverse events (irAEs) and even cytokine release syndrome (CRS), and these potential systemic toxicities have severely limited its clinical application.

[0006] Against this background, the core technical problem in this field is: how to develop a novel treatment method that can effectively kill tumor cells while safely and efficiently activating and reshaping the tumor immune microenvironment, thereby overcoming the limitations of single therapy. Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant oncolytic adenovirus expressing IL-36, its construction method, and its applications. The aim is to create a novel recombinant oncolytic adenovirus H101+IL-36 by inserting the IL-36 gene into the genome of oncolytic adenovirus H101. This recombinant virus can specifically express IL-36 during tumor cell infection, thereby enhancing the local tumor immune response and significantly increasing CD8+ expression. + T cells and dendritic cells function together to produce a more significant anti-tumor immune response.

[0008] The objective of this invention can be achieved through the following technical solutions: A method for constructing a recombinant oncolytic adenovirus expressing IL-36, comprising the following steps: S1. The human IL-36G gene was inserted into the adenovirus shuttle plasmid through the Xba I and Bgl II restriction sites to obtain the recombinant shuttle plasmid. S2. After linearizing the recombinant shuttle plasmid by Pme I enzyme digestion, it was co-electrotransformed with the adenovirus backbone plasmid into competent cells for homologous recombination. Positive clones were picked and extracted to obtain the recombinant adenovirus plasmid. S3. After linearizing the recombinant adenovirus plasmid by Pac I enzyme digestion, it was transfected into packaging cells using transfection agent. After 10 days of transfection, obvious cytopathic effect was observed. The virus fluid was collected and subjected to three freeze-thaw cycles to obtain the primary virus stock solution. S4. Infect packaging cells with the primary virus stock solution for amplification. After the cells are fully cytopathic, collect the virus solution and purify it using cesium chloride density gradient centrifugation. After purification, store it in PBS buffer containing 10% glycerol at -80°C to obtain recombinant oncolytic adenovirus expressing IL-36.

[0009] Furthermore, the NCBI Gene ID of the human IL-36G gene is 56300.

[0010] Furthermore, the adenovirus shuttle plasmid is derived from the pShuttle-CMV shuttle plasmid produced by Agilent Technologies, and its multiple cloning site contains multiple restriction endonuclease sites, which facilitates the insertion of foreign genes.

[0011] Furthermore, the adenovirus backbone plasmid is pAdEasy-1, which has the same serotype and core genome structure as oncolytic adenovirus H101.

[0012] Furthermore, the competent cells are BJ5183, which are Escherichia coli competent cells, and have a high homologous recombination efficiency, which can effectively promote the formation of recombinant plasmids.

[0013] Furthermore, the packaging cells are HEK293A, which are derived from ATCC CRL-1573 and can efficiently support adenovirus replication and packaging.

[0014] Furthermore, the specific operation of the cesium chloride density gradient centrifugation method includes: firstly, centrifuging at 3000×g for 10 min to remove cell debris, filtering the supernatant through a 0.45μm filter membrane, adding a cesium chloride solution with a final concentration of 1.34g / mL, and ultracentrifuging at 28000rpm and 4℃ for 2 h to collect the virus bands, and using a desalting column to remove cesium chloride.

[0015] A recombinant oncolytic adenovirus expressing IL-36 was prepared by the above construction method. This recombinant oncolytic adenovirus carries the IL-36 gene and can specifically express IL-36 during the infection of tumor cells.

[0016] The application of a recombinant oncolytic adenovirus expressing IL-36 in the treatment of tumors can improve the efficacy of tumor treatment, especially for colorectal cancer.

[0017] Compared with the prior art, the present invention has the following significant advantages and beneficial effects: 1. Synergistic Anti-tumor Immune Activation: This invention organically combines the direct oncolytic effect of oncolytic viruses with the immunomodulatory function of IL-36. The virus lyses tumor cells, releasing tumor-associated antigens and danger signals, while locally expressed IL-36 synergistically promotes dendritic cell (DC) maturation and enhances CD8+. + T cell cytotoxicity drives tumor-associated macrophages (TAMs) to polarize towards the anti-tumor M1 phenotype, thereby effectively reversing the immunosuppressive "cold" TME into an immune-active "hot" TME.

[0018] 2. High safety and low off-target effects: The oncolytic virus vector specifically expresses IL-36 locally in the tumor, achieving targeted delivery of cytokines and significantly reducing the potential toxic side effects of systemic IL-36 application. Adenovirus H101 itself has good tumor targeting properties, further reducing damage to normal tissues.

[0019] 3. Significant anti-tumor effects: In vitro experiments have demonstrated that recombinant oncolytic adenovirus can effectively inhibit the activity of colon cancer cells and induce apoptosis. In vivo animal experiments have confirmed that intratumoral injection of recombinant oncolytic adenovirus can significantly inhibit tumor growth, reduce tumor weight, and prolong the survival of tumor-bearing mice. Attached Figure Description

[0020] The invention will now be further described with reference to the accompanying drawings.

[0021] Figure 1 This is a diagram showing the staining morphology results of cell apoptosis and death detected using the DAPI and SYTOX Green nucleic acid dye double staining method in Experiment Example 1 of this invention. Figure 2 This is a graph showing the quantitative statistical results of cell apoptosis and death detected by the DAPI and SYTOX Green nucleic acid dye double staining method in Experiment Example 1 of this invention; Figure 3 This is a graph showing the results of cell viability detection using the CCK-8 assay in Experiment Example 1 of this invention; Figure 4 This is a comparison chart of the results of detecting the expression of key signaling pathway proteins using Western blot in Experiment Example 2 of this invention; Figure 5 This is a statistical chart of the final weight of the transplanted tumor in mice in Experiment Example 4 of this invention. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1 This embodiment provides a recombinant oncolytic adenovirus expressing IL-36, which is prepared by the following method: S1. The human IL-36G gene (NCBI Gene ID: 56300) was inserted into the multiple cloning site of the pShuttle-CMV shuttle plasmid (produced by Agilent Technologies) through the Xba I and Bgl II restriction sites to obtain the recombinant shuttle plasmid, denoted as pShuttle-CMV-IL-36. S2. After linearizing the recombinant shuttle plasmid pShuttle-CMV-IL-36 by Pme I restriction enzyme, it was co-electrotransformed with the pAdEasy-1 backbone plasmid into BJ5183 competent cells for homologous recombination. Positive clones were picked and extracted to obtain the recombinant adenovirus plasmid, denoted as pAdEasy-IL-36. S3. After linearizing the recombinant adenovirus plasmid pAdEasy-IL-36 by Pac I enzyme, it was transfected into HEK293A packaging cells (ATCC CRL-1573) using Lipofectamine 3000 transfection agent. After 10 days of transfection, obvious cytopathic effect was observed. The virus fluid was collected and subjected to three freeze-thaw cycles to obtain the primary virus stock solution. S4. The primary viral stock solution was used to infect HEK293A packaging cells for amplification. After the cells were fully cytopathic, the viral solution was collected and purified using the cesium chloride density gradient centrifugation method: First, the cells were centrifuged at 3000×g for 10 min to remove cell debris. The supernatant was filtered through a 0.45μm filter membrane, and a cesium chloride solution with a final concentration of 1.34g / mL was added. The cells were then ultracentrifuged at 28000rpm and 4℃ for 2 h. The viral bands were collected, and the cesium chloride was removed using a desalting column (PD-10, GE Healthcare). After purification, the virus was stored in PBS buffer containing 10% glycerol at -80℃ to obtain a recombinant oncolytic adenovirus expressing IL-36, denoted as H101+IL-36.

[0024] TCID 50 The viral titer was determined to be 2.5 × 10⁻⁶. 10 TCID 50 / mL. HCT-116 cells (ATCC CCL-247) were infected with H101+IL-36 (MOI=10). After 72 h, cell supernatant and lysate were collected. Western blot analysis showed a distinct IL-36-specific band at approximately 18 kDa. ELISA analysis showed that the secretion of IL-36 in the cell supernatant reached 125.6 ± 8.7 ng / mL. 6 Cells / 24h; both Western blot and ELISA confirmed high levels of IL-36 protein expression in cell supernatant and lysate.

[0025] Experimental Example 1: Evaluation of in vitro antitumor effects Human colon cancer cell lines HCT-116 (ATCC CCL-247) and SW480 (ATCC CCL-228) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). The experiment was conducted in four groups: ① PBS control group; ② H101 single drug group (MOI=10); ③ IL-36 single drug group (100 ng / mL); ④ H101 + IL-36 treatment group (MOI=10).

[0026] Cell apoptosis and cell death were detected using a double staining method with DAPI and SYTOX Green nucleic acid dyes. DAPI labeled the nuclei of all cells, producing strong blue fluorescence, while SYTOX Green could only penetrate into late-stage apoptotic and necrotic cells with damaged cell membranes, emitting green fluorescence. After 48 hours of treatment, observation and counting were performed using a fluorescence microscope or a high-content imaging system. Figure 1 It can be seen that a large number of SYTOX Green-positive cells (late apoptosis / necrosis) appeared in the H101+IL-36 treatment group, while the cell nuclei in the PBS control group were only stained with DAPI (live cells); Figure 2 It can be seen that the cell death rate induced by the H101+IL-36 treatment group was significantly higher than that of the other groups; Cell viability was detected using the CCK-8 assay: cells were sputtered at a concentration of 5 × 10⁻⁶. 3 Cells were seeded in 96-well plates and treated for 24, 48, and 72 hours. Then, 10 μL of CCK-8 reagent (Dojindo) was added to each well, and after incubation for 2 hours, the absorbance was measured at 450 nm. Cell viability (%) was calculated as follows: (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%. Figure 3 The results showed that, compared with the PBS control group, the H101 monotherapy group, or the IL-36 monotherapy group, the H101+IL-36 treatment group had the lowest survival rate against human colon cancer cells HCT-116 and SW480, effectively inhibiting the proliferation of colon cancer cells.

[0027] Experiment Example 2: Signal Path Analysis Western blot analysis was used to analyze the expression of proteins in key signaling pathways. Results are as follows: Figure 4 As shown, compared with single treatment (H101 or IL-36), H101+IL-36 treatment can downregulate the key protein of the Wnt / β-catenin pathway (β-catenin, TCF7), while enhancing the phosphorylation of NF-κB p65 and significantly increasing the level of Cleaved Caspase-3, suggesting that it exerts its anti-tumor effect through multi-pathway synergy.

[0028] Experiment Example 3: Effects on Macrophage Polarization Preparation of Tumor-Conditioned Medium (TCM) To investigate the effects of secretome on the immune microenvironment after co-culturing tumor cells with oncolytic viruses / cytokines, the inventors collected conditioned medium from tumor cells. The specific steps are as follows: HCT-116 or SW480 cells in logarithmic growth phase were seeded at an appropriate density in 6-well plates. After cell attachment, the original culture medium was discarded and replaced with fresh RPMI-1640 medium containing 2% FBS. Cells were then treated according to the groups (①PBS control group; ②H101 single-drug group; ③IL-36 single-drug group; ④H101+IL-36 treatment group). Cells were cultured at 37℃ in a 5% CO2 incubator for 48 h. After culture, the cell supernatant was carefully collected from each well, avoiding aspirating cell debris. The collected supernatant was centrifuged at 2000×g for 10 min at 4℃ to thoroughly remove suspended cells and debris. The centrifuged supernatant was filtered through a sterile syringe filter with a 0.45 μm pore size for sterilization. The filtered TCM was aliquoted into sterile centrifuge tubes and stored at -80℃ for long-term use.

[0029] THP-1 cells (ATCC TIB-202) were used at a rate of 5 × 10⁻⁶. 5 The cells were resuspended at a density of cells / mL in RPMI-1640 complete medium containing 10% FBS. Phorbol ester (PMA) was added to the medium to a final concentration of 100 ng / mL to induce THP-1 cells to differentiate into adherent M0 macrophages. Cells were seeded in 6-well plates or culture dishes and incubated at 37°C in a 5% CO2 incubator for 24 h.

[0030] After induction, the culture medium containing PMA was discarded, and the adherent cells were gently washed twice with pre-warmed PBS to thoroughly remove undifferentiated cells and PMA residue. The medium was then replaced with fresh complete culture medium, and the cells were allowed to rest for 24 hours to obtain mature M0 macrophages.

[0031] M0 macrophages were randomly grouped and cultured in TCM medium (TCM and fresh medium mixed at a 1:1 volume ratio) for 24 h to induce polarization. Total RNA was extracted from macrophages in each group using TRIzol reagent (Invitrogen), and then 1 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, PrimeScript™ RT reagent Kit). qPCR reactions were performed on a QuantStudio real-time quantitative PCR system (Applied Biosystems) using SYBR Green premixed reagent (TaKaRa, TB Green® Premix Ex Taq™). Genes detected included: M1 biomarkers—inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12); and M2 biomarkers—arginase-1 (Arg-1), CD206, and interleukin-10 (IL-10).

[0032] Flow cytometry detection of M1 surface markers: Macrophages treated with TCM were carefully collected using a cell scraper, and single-cell suspensions were prepared. Cells were resuspended in PBS (FACS Buffer) containing 2% FBS, and anti-human CD86 antibody (BioLegend, clone: ​​IT2.2) or its isotype control antibody was added. The cells were incubated at 4°C in the dark for 30 min. After incubation, the cells were washed twice with FACS Buffer and finally resuspended in 300 μL of buffer. Flow cytometry was used for detection, and the proportion of CD86-positive cells was analyzed using FlowJo software.

[0033] qRT-PCR and flow cytometry results showed that TCM in the H101+IL-36 treatment group significantly upregulated the expression of M1 markers (iNOS, TNF-α, CD86) while downregulating the expression of M2 markers (Arg-1, CD206), indicating that it can effectively drive macrophages to polarize towards the anti-tumor M1 phenotype.

[0034] Experiment Example 4: Evaluation of in vivo antitumor effects Subcutaneous xenografts were established in C57BL / 6 mice using MC38 colon cancer cells. The tumors were then intratumorally injected with equal volumes of PBS, H101, and IL-36, or recombinant oncolytic adenovirus H101+IL-36. Tumor volume was measured periodically. The animal experimental methods are as follows: Six-week-old female C57BL / 6 mice were selected, and each mouse was injected with 5 × 10⁻⁶ mg / L. 5MC38 cells were injected subcutaneously at a rate of 100 μL / cell into the right axilla. Tumor growth was observed every two days after injection, and the tumor volume (mm²) was calculated using digital calipers. 3 = (length × width) 2 ) / 2. Wait until the tumor volume grows to approximately 50-100 mm. 3 Mice were then divided into four groups: PBS group, H101 group, IL-36 group, and H101+IL-36 group, with 6-8 mice in each group. After grouping, each mouse was injected intratumorally with 1×10⁻⁶ PBS. 8 PFU virus or an equal volume of PBS was injected twice a week for a total of four times, and then the weight and volume changes of the tumor were measured regularly.

[0035] The results are as follows Figure 5 As shown, compared with the PBS group, tumor growth in mice treated with H101+IL-36 was significantly inhibited, and the tumor weight was ultimately significantly reduced. Throughout the experiment, the weight of the mice in the treatment group remained stable, and no obvious toxic reactions were observed, indicating that H101+IL-36 has good in vivo safety and significant anti-tumor efficacy.

[0036] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0037] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method of constructing a recombinant oncolytic adenovirus expressing IL-36, characterized in that, Comprise the following steps: S1, the human IL-36G gene is inserted into the adenovirus shuttle plasmid through Xba I and Bgl II enzyme cutting site, and the recombinant shuttle plasmid is obtained; S2, the recombinant shuttle plasmid is linearized by Pme I enzyme cutting, and then co-electrotransferred into competent cells with adenovirus backbone plasmid to carry out homologous recombination, and the positive clone is picked and extracted, to obtain the recombinant adenovirus plasmid;S3, the recombinant adenovirus plasmid is linearized by Pac I enzyme cutting, and then transfected into packaging cells using transfection agent, and obvious cytopathic effect is observed after 10 days of transfection, the virus liquid is collected and subjected to repeated freeze-thawing three times, and the primary virus stock solution is obtained; S4, the primary virus stock solution is infected to packaging cells for amplification, and the virus liquid is collected after the cells are completely pathogenic, and the cesium chloride density gradient centrifugation method is used for purification, and the purified product is stored in PBS buffer containing 10% glycerol, and stored at-80℃, to obtain the recombinant oncolytic adenovirus expressing IL-36.

2. The method of constructing a recombinant oncolytic adenovirus expressing IL-36 according to claim 1, wherein, The NCBI Gene ID of the human IL-36G gene is 56300.

3. The method of constructing a recombinant oncolytic adenovirus expressing IL-36 according to claim 1, wherein, The adenovirus shuttle plasmid is derived from the pShuttle-CMV shuttle plasmid produced by Agilent Technologies company.

4. The method of constructing a recombinant oncolytic adenovirus expressing IL-36 according to claim 1, wherein, The adenovirus backbone plasmid is pAdEasy-1.

5. The method of constructing a recombinant oncolytic adenovirus expressing IL-36 according to claim 1, wherein, The competent cell is BJ5183.

6. The method of constructing a recombinant oncolytic adenovirus expressing IL-36 according to claim 1, wherein, The packaging cell is HEK293A.

7. The method of constructing a recombinant oncolytic adenovirus expressing IL-36 according to claim 1, wherein, The specific operation of the cesium chloride density gradient centrifugation method comprises the following steps: firstly, remove the cell fragments by centrifugation at 3000xg for 10min, filter the supernatant through a 0.45μm filter membrane, then add cesium chloride solution with a final concentration of 1.34g / mL, and centrifuge at 28000rpm and 4℃ for 2h, collect the virus band, and remove the cesium chloride using a desalting column.

8. A recombinant oncolytic adenovirus expressing IL-36, characterized in that, Prepared by the construction method of any one of claims 1-7.

9. The recombinant oncolytic adenovirus expressing IL-36 of claim 8 in the treatment of tumors.

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