A recombinant oncolytic virus targeting CD317 gene and application thereof in anti-tumor

Abstract

The application discloses a recombinant oncolytic virus targeting CD317 gene and application thereof in anti-tumor, and belongs to the technical field of tumor treatment. The recombinant oncolytic virus comprises a CD317 inhibitor and an oncolytic virus, and is formed by integrating the CD317 inhibitor into the oncolytic virus genome. The CD317 inhibitor is a substance capable of inhibiting CD317 gene expression or targeting degradation of CD317 protein function, and is selected from shRNA or siRNA targeting CD317. The application develops the oncolytic virus targeting knockdown of CD317 expression, inhibits tumor cell proliferation by reducing CD317 expression of tumor cells, reduces PD-L1 expression so as to break the immune escape mechanism, simultaneously enhances the killing sensitivity of tumor cells to CD8+ T cells, forms a synergistic effect with the oncolysis of the oncolytic virus, and the recombinant oncolytic virus has stronger in-vivo anti-tumor activity, thereby providing a new potential scheme for CD317-driven tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of tumor treatment technology, specifically relating to a recombinant oncolytic virus targeting the CD317 gene and its application in anti-tumor therapy. Background Technology

[0002] Cancer is a non-hereditary genetic disease. Normal cells, under the influence of carcinogenic factors, undergo gene alterations, losing their normal regulation of growth and leading to abnormal proliferation. Tumor cells have three significant basic characteristics: immortality, migration, and loss of contact inhibition. The core idea of ​​cancer treatment is to kill and eliminate tumor cells. Currently, commonly used clinical cancer treatments include chemotherapy, radiotherapy, and hormone therapy. However, almost all drugs develop resistance in cancer cells over time, causing the drugs to cease their effect. This acquired resistance of tumor cells to drug treatment not only severely limits the effectiveness of clinical treatment but is also the molecular basis for tumor recurrence. Therefore, we need to develop new treatment methods or optimize existing ones. On the one hand, this can break drug resistance and enhance anti-tumor effects; on the other hand, it can enrich our "arsenal" against cancer and broaden the range of cancer treatment strategies.

[0003] Oncolytic virus therapy is a rapidly developing innovative strategy in the field of cancer treatment in recent years. Traditional oncolytic viruses mainly rely on their selective replication and direct oncolytic effect within tumor cells, resulting in limited clinical efficacy. To overcome this bottleneck, a new generation of recombinant oncolytic viruses has emerged. These viruses are engineered by inserting exogenous therapeutic genes into their genome, enabling them to express specific cytokines, immunomodulatory factors, or other functional elements after infecting tumor cells, thereby enhancing their anti-tumor activity or endowing them with new therapeutic properties. This strategy has been proven effective by numerous studies. For example, modified oncolytic vaccinia viruses can express immune factors such as IL-2, IL-15, or HBD2, thereby more precisely and effectively identifying and eliminating tumor cells. Studies have also observed stronger anti-tumor effects in solid tumor models by expressing bacterial flagellin (a TLR5 ligand) in recombinant vaccinia viruses. The design of the third-generation oncolytic virus VG161 is particularly typical: it not only expresses IL-12 and IL-15 to synergistically activate T cells and NK cells, but also secretes a PD-L1 blocking peptide to reverse the immunosuppressive microenvironment of the tumor. This "dual immune protection" design ensures that it can trigger a stronger and more systematic anti-tumor immune response in vivo. These achievements not only establish the technical path and feasibility of targeted modification, but also form a core consensus: discovering and targeting key immune regulatory nodes or functional genes is a potential solution to improve the efficacy of oncolytic viruses.

[0004] CD317, also known as bone marrow stromal antigen 2 (BST-2), tetherin, or HM1.24, is a type II transmembrane glycoprotein with a molecular weight of 29-33 kDa. It is a surface molecule overexpressed in tissues and plasma samples from multiple myeloma, B-lymphoma, glioblastoma multiforme, primary lung cancer, head and neck squamous cell carcinoma, endometrial cancer, brain cancer, breast cancer with bone metastases, chronic B-lymphoblastic leukemia, and colorectal cancer. It also specifically upregulates cancer cells in metastatic tumor cells and chemotherapy-resistant cells. CD317 overexpression is not only positively correlated with clinical indicators such as poor tumor prognosis, but has also been shown to directly promote tumor development and progression in various cell and animal models.

[0005] CD317 is a potential target for cancer therapy, but a mature application system is currently lacking. Existing CD317-targeted therapies mainly include antibodies and peptides. Antibodies were the earliest strategy attempted to target CD317 for cancer treatment, but their over-reliance on ADCC effects limits their potential application in patients with advanced cancer, and there have been no successful precedents to date. Although CD317 intervention peptides have been shown to block cancer cell adhesion and proliferation, their efficacy in preclinical models is severely insufficient, and the instability of peptides also limits the further development and utilization of this strategy. Our recent research found that CD317 not only promotes cancer cell proliferation but also upregulates PD-L1 expression, promoting immune escape. Overexpression of CD317 in hepatocellular carcinoma enhances EGFR activation through a lipid valve-dependent mechanism, thereby promoting liver cancer cell proliferation. Knocking down CD317 in cancer cells using siRNA or shRNA can significantly inhibit cancer cell proliferation, reduce PD-L1 expression, and thus enhance their sensitivity to T cell killing.

[0006] Based on this, it is necessary to develop a method that can simultaneously block the proliferative and immune escape regulatory effects of CD317 and form a synergistic therapy with oncolytic viruses. Therefore, this invention combines a CD317 inhibition strategy with an oncolytic virus platform to develop an oncolytic virus that targets and knocks down CD317 expression. It has been demonstrated that this recombinant oncolytic virus has stronger in vivo antitumor activity, providing a new potential approach for CD317-driven tumor therapy. Summary of the Invention

[0007] In view of the technical defects of existing oncolytic viruses with limited efficacy and CD317-targeting antibody or peptide therapy with limited application, the present invention provides a technical solution of combining or recombining CD317 inhibitors with oncolytic viruses, and discloses a recombinant oncolytic virus targeting the CD317 gene and its application in anti-tumor treatment.

[0008] This invention mainly relates to the combined use of CD317-targeting shRNA with traditional oncolytic viruses or the integration of CD317 shRNA into the genome of oncolytic viruses to form recombinant oncolytic viruses. The mechanism of action is to reduce the expression of CD317 in cancer cells, thereby inhibiting cancer cell proliferation, and to reduce PD-L1 expression, thereby breaking the immune escape mechanism and enhancing the anti-tumor efficacy of recombinant oncolytic viruses used alone or in combination with T-cell therapy.

[0009] The first aspect of the present invention discloses a recombinant oncolytic virus comprising a CD317 inhibitor and an oncolytic virus, wherein the recombinant oncolytic virus is formed by integrating the CD317 inhibitor into the genome of the oncolytic virus; wherein the CD317 inhibitor is a substance capable of inhibiting CD317 gene expression or targeting the degradation of CD317 protein.

[0010] Furthermore, the CD317 inhibitor is selected from shRNA or siRNA that targets CD317, and its mechanism of action is to inhibit CD317 gene expression.

[0011] Furthermore, the oncolytic virus is an oncolytic adenovirus, herpes simplex virus, vaccinia virus, Newcastle disease virus, parvovirus, measles virus, or poliovirus, preferably an oncolytic adenovirus.

[0012] A second aspect of the present invention discloses a pharmaceutical composition, characterized in that it comprises the recombinant oncolytic virus as described in any one of claims 1-3 and a pharmaceutically acceptable vector.

[0013] The third aspect of this invention discloses the use of the recombinant oncolytic virus or the pharmaceutical composition in the preparation of antitumor drugs.

[0014] Furthermore, in the aforementioned application, the tumor is a tumor that highly expresses CD317.

[0015] Furthermore, in the aforementioned application, the tumor is selected from liver cancer and breast cancer.

[0016] Furthermore, in the aforementioned application, the recombinant oncolytic virus or drug composition inhibits cancer cell proliferation by reducing CD317 expression in cancer cells and breaks the immune escape mechanism by reducing PD-L1 expression.

[0017] Furthermore, in the aforementioned application, the antitumor drug can be used in combination with T-cell immunotherapy drugs.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The combined application of the CD317 inhibitor (mainly shRNA or siRNA) and oncolytic virus involved in this invention has a significantly better tumor-suppressing effect than a single agent, and can significantly inhibit tumor progression, providing a new candidate for clinical treatment.

[0019] The recombinant oncolytic virus of this invention can also downregulate the expression of the immune checkpoint PD-L1, and has the potential to enhance the anti-tumor effects of T cell-based immunotherapies (such as anti-PD-L1, CAR-T, and TIL). It can increase the sensitivity of tumor cells to killing CD8+ T cells, forming a synergistic effect with the immune activation of oncolytic viruses. Attached Figure Description

[0020] Figure 1 The results show the effects of CD317 on tumor cell proliferation and clonogenic ability. Figure A shows the HCT116 cell proliferation curve, where CD317 overexpression significantly promotes cell proliferation. Figure B shows the results and statistics of clonogenic ability detection in different tumor cells, showing that CD317 overexpression significantly improves clonogenic ability in HCT116 colon cancer cells, Huh7 liver cancer cells, MDA-MB-468 breast cancer cells, and MCF7 breast cancer cells. Figure C shows the results and statistics of clonogenic ability detection in RKO cells, where the clonogenic ability of CD317 knocked-down cancer cells (sh144, sh452) is reduced compared with control cells (PLVTHM).

[0021] Figure 2 The figures show the results of CD317 overexpression promoting xenograft growth. Figure A is the xenograft growth curve, showing that CD317 overexpression promotes xenograft growth. Figure B is the actual xenograft at the experimental endpoint, showing that the xenograft in the CD317 overexpression group is significantly larger than that in the control group. Figure C is the xenograft weight statistics, showing that the xenograft in the CD317 overexpression group is heavier than that in the control group.

[0022] Figure 3 The figures show the results of CD317 knockdown and overexpression regulating PD-L1 protein expression levels. Figure A shows a Western blot of CACO2 cells, indicating that CD317 knockdown (sh144, sh405) significantly inhibits PD-L1 expression; Figure B shows a Western blot of RKO cells, indicating that CD317 knockdown significantly inhibits PD-L1 expression; Figure C shows a Western blot of MDA-MB-468 cells, indicating that CD317 overexpression promotes PD-L1 expression; Figure D shows a Western blot of HCT116 cells, indicating that CD317 overexpression significantly promotes PD-L1 expression; and Figure E shows a Western blot of MCF7 cells, indicating that CD317 overexpression significantly promotes PD-L1 expression.

[0023] Figure 4The results show the effects of CD317 knockdown or overexpression on the regulation of PD-L1 levels in cancer cell membranes. Figure A shows the flow cytometry analysis of PD-L1 in CACO2 cells, where PLVTHM is the control cell, and sh144 and sh452 are CD317 knockdown cells. It can be seen that CD317 knockdown reduces the level of PD-L1 on the cell surface. Figure B shows the flow cytometry analysis of PD-L1 in HCT116 cells, where PLVX is the control cell and CD317 is the overexpressing cell. It can be seen that CD317 overexpression significantly increases the level of PD-L1 on the cell membrane. Figure C shows the flow cytometry analysis of PD-L1 in Huh7 cells, where PLVX is the control cell and CD317 is the overexpressing cell. To more intuitively observe the effect of CD317 on PD-L1 expression, the positive (Positive, Pos) and negative (Negative, Neg) populations within the CD317 overexpression group (distinguished by the co-expressed gene GFP) were analyzed separately. The results showed that CD317 overexpression significantly increased the level of PD-L1 on the cell membrane.

[0024] Figure 5 Knock down CD317 to enhance CD8 + The results of the T cell anti-tumor effect are shown in Figure A, which is a representative flow cytometry analysis of RKO cells and T cells after co-culture, where CD8-GFP+ represents tumor cells and CD8+GFP- represents CD8+ T cells; Figure B shows the statistical analysis results of the percentage of CD8+ T cells in the co-culture system, indicating that the proportion of CD8+ T cells in the CD317 knockdown group (sh144) is higher; Figure C shows the statistical analysis results of the proportion of tumor cells in the co-culture system, showing that the number of cancer cells in the CD317 knockdown group is significantly lower than that in the control group.

[0025] Figure 6 Figure A shows the design and validation results of oncolytic viruses targeting CD317. Figure A shows the schematic diagram of the oncolytic adenovirus vector structure and the sequencing results of the CD317 shRNA sequence inserted therein. Figure B shows the validation of the effectiveness of the CD317 shRNA used for the construction of oncolytic adenovirus.

[0026] Figure 7 Figure 1 shows the results of CD317 shRNA oncolytic virus significantly inhibiting the growth of RKO xenografts. Figure 2 shows the volume monitoring of xenografts in mice, indicating that CD317 shRNA oncolytic adenovirus (OV-shCD317) significantly inhibits the growth of xenografts. Figure 3 shows the statistical analysis results of xenograft tumors at the experimental endpoint, indicating that the tumor weight in the OV-shCD317 treatment group was smaller than that in the control group (OV-shCT). Figure 4 shows a representative xenograft at the experimental endpoint. Detailed Implementation

[0027] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0028] The present invention will be described in detail below through specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the experimental materials used are all commercially available products.

[0029] Example 1: Effects of CD317 on tumor cell proliferation and clonogenic ability I. Construction of Stable Cell Lines 1. Lentiviral Packaging: HEK 293T cells were plated into 10cm culture dishes one day in advance. Once the cell confluence reached 80%, lentivirus packaging could begin. Take a 1.5mL centrifuge tube, add 1mL of Opti MEM, and then add the target vector, psPAX2, and pMD2G in a 4:3:1 ratio to the centrifuge tube. Mix well, then add 60μL of PEI, vortex for 15s, and incubate at room temperature for 15min. Slowly add the prepared transfection complex to the culture dish, changing the medium every 6-8 hours.

[0030] 2. Virus Collection and Concentration: 48 h after transfection, collect the cell supernatant and centrifuge at 1000g for 5 min to remove cell debris. Mix the supernatant with the virus concentrate at a ratio of 4:1 and incubate overnight at 4°C on a shaker. After incubation, centrifuge at 4000g for 30 min, discard the supernatant, and resuspend the pellet in PBS.

[0031] 3. Target cell infection: Target cells were seeded into 24-well plates, concentrated virus solution was added, and polybrene was added at a ratio of 1:1000. After 24 hours of infection, the medium was replaced with fresh medium. After 72 hours of infection, a portion of the cells was taken for flow cytometry to detect the infection efficiency.

[0032] II. Effects of MTS detection on CD317 overexpression on tumor cell proliferation HCT116 cells stably expressing CD317 and Vector were loaded with 3 × 10⁻⁶ cells. 3 Cells were seeded at a density of 6 replicates per group in 96-well plates, with a blank well containing only culture medium. The 96-well plates were incubated in a CO2 incubator for 24 hours to allow cell adhesion. At 24, 48, 72, and 96 hours post-seeding, 20 μL of MTS reagent was added to each well, and incubation continued for another 4 hours. The absorbance of each well was measured at 490 nm using a microplate reader. III. The Influence of CD317 on Tumor Cell Clone Formation Ability as Detected by Plate Cloning Prepare single-cell suspensions of CD317 overexpressing or knockdown cells and their corresponding control cells, and seed the cells at a density of 1000 cells / well in 6-well plates. Incubate the 6-well plates at 37°C in a 5% CO2 incubator, changing the medium with fresh medium every 2-3 days. Stop the culture when visible clones appear. After culture, discard the old medium and wash twice with PBS buffer.

[0033] Add 1 mL of 4% paraformaldehyde to each well and fix at room temperature for 30 min. Discard the fixative and wash twice with PBS buffer. Add 1 mL of crystal violet staining solution to each well and stain for 30 min. Recover the crystal violet staining solution, rinse slowly with running water, and invert the 6-well plate to air dry. Take photos and record the data. Use ImageJ software to count the number of clones.

[0034] like Figure 1 The figure shows the effect of CD317 on tumor cell proliferation and colony formation. It can be seen from the figure that CD317 overexpression significantly promotes the proliferation of HCT116 cells (MSI). Figure 1 A) and clone-forming ability ( Figure 1 B), also significantly enhanced the clonogenic ability of Huh7 liver cancer cells, MDA-MB-468 breast cancer cells, and MCF7. Figure 1 B). Conversely, knockdown of CD317 expression in RKO cells significantly inhibited the clonogenic ability of cancer cells (B). Figure 1 C). These results indicate that CD317 directly promotes cancer cell proliferation and colony formation, providing a core target basis for CD317-targeted tumor therapy.

[0035] Example 2: CD317 overexpression promotes xenograft growth Collect HCT116-CD317-OE cells and HCT116-Vector cells in the logarithmic growth phase and adjust the cell concentration to 5 × 10⁻⁶. 7 Cells / mL. Female NSG mice aged 6-8 weeks were randomly divided into two groups of 5 mice each. Each mouse was subcutaneously injected with 100 μL of cell suspension. Starting on day 7 post-inoculation, the long axis (L) and short axis (W) of the transplanted tumor were measured every two days using calipers. The tumor volume V was calculated as V = (L × W) / (L × W). 2 () / 2, and plot the growth curve. When the tumor volume reaches approximately 1500 mm... 3 At that time, all mice were euthanized, the tumor tissue was removed and weighed, and the difference in tumor weight between the two groups was compared.

[0036] like Figure 2 The image shows the results of CD317 overexpression promoting xenograft growth. The research team constructed a mouse xenograft model of HCT116 and found that CD317 overexpression significantly promoted xenograft growth. Figure 2 A). At the experimental endpoint, the xenografts in the overexpression group were significantly larger than those in the control group ( Figure 2 BC).

[0037] Example 3: The regulatory effect of CD317 on PD-L1 expression I. Western blot analysis of the effect of CD317 on PD-L1 expression 1. Cell treatment 1) The tumor cells were divided into 6×10 5 Cells / well were seeded at a density in 6-well plates. 2) When the cell confluence reaches 50-60%, transfect PLVX, PLVX-CD317, CD317-siRNA and control siRNA respectively.

[0038] 3) Collect cells 48 hours after transfection.

[0039] 2. Western blot detection of PD-L1 expression 1) Wash cells with pre-chilled PBS buffer, add 100 μL of RIPA lysis buffer containing protease inhibitors to each well, and lyse on ice for 30 min. Centrifuge at 13200 rpm for 15 min and collect the supernatant. Add 5× loading buffer to the protein sample and heat in a metal bath at 100°C for 10 min.

[0040] 2) Take protein samples for SDS-PAGE electrophoresis. After electrophoresis, transfer the proteins to a PVDF membrane using a wet transfer method. 3) Place the PVDF membrane in 5% BSA and block at room temperature for 2 hours. After blocking, wash the membrane three times with PBST buffer, 5 minutes each time.

[0041] 4) Add CD317, PD-L1, and β-actin antibodies respectively, and incubate overnight at 4°C. The next day, wash the membrane three times with PBST buffer, 5 min each time. Add HRP-labeled secondary antibody, incubate at room temperature for 2 h, and wash the membrane three times with PBST, 10 min each time after incubation.

[0042] 5) Add the ECL chemiluminescent reagent evenly onto the membrane and then develop it.

[0043] like Figure 3 The figure shows the results of CD317 knockdown or overexpression regulating PD-L1 protein expression levels. As can be seen from the figure, in the colon cancer cell lines CACO2 and RKO, CD317 knockdown also significantly reduced PD-L1 expression. Figure 3 A, B). Overexpression of CD317 in MDA-MB-468, HCT116, and MCF7 cells significantly increased PD-L1 protein levels (A, B). Figure 3 (CE), further confirming the role of CD317 in regulating PD-L1 expression.

[0044] II. Effect of CD317 on membrane PD-L1 expression detected by flow cytometry Collect the cell suspension and wash the cells once with pre-chilled PBS buffer. Add CD317 antibody and incubate at 4°C in the dark for 20 min. After incubation, wash with PBS buffer to remove unbound antibody. Resuspend the cells in 100 μL PBS, add 1 μL of APC-anti-mouse IgG and PE-PD-L1 antibody to each tube, and incubate at 4°C in the dark for 20 min. Wash with PBS. Resuspend the cells in 200 μL PBS and detect CD317 and PD-L1 expression by flow cytometry.

[0045] like Figure 4 The figure shows the results of CD317 knockdown or overexpression regulating PD-L1 levels in cancer cell membranes. As can be seen from the figure, PD-L1 mainly promotes tumor immune escape by binding to PD-1 and transmitting inhibitory signals, and its level on the cell membrane is particularly important for this function. Therefore, we also used flow cytometry to analyze the effect of CD317 on PD-L1 levels in tumor cell membranes. The results are consistent with the previous results; CD317 knockdown significantly reduced PD-L1 levels on the surface of CACO2 cells (…). Figure 4 A), while CD317 overexpression promotes PD-L1 expression on the cell membrane ( Figure 4 B, C).

[0046] Example 4: Flow cytometry detection of the effect of CD317 on T cell killing sensitivity 1. Activation of primary human T cells Take PBMCs that have recovered their activity after resuscitation, add anti-CD3 / CD28 activation magnetic beads to the PBMCs at a ratio of 3:1 of magnetic beads to cells, gently mix by pipetting, and activate for 48 hours.

[0047] 2. Establishment of a co-training system RKO cells from the CD317 knockdown group and the control group were seeded with activated T cells at an E:T ratio of 1:1 in 96-well plates, with a total cell count of 2 × 10⁶ cells. 4 Each group was divided into 3 replicates and incubated in a 37℃, 5% CO2 incubator for 24 hours.

[0048] 3. Flow cytometry detection of CD8 + ratio of T cells to tumor cells After co-culture, cell suspensions were collected from each well, and cells were washed with PBS buffer. Cells were resuspended in 100 μL PBS, and 1 μL of PE-CD8 antibody was added to each tube. The cells were incubated at 4°C in the dark for 20 min, and unbound antibody was washed away with PBS. Cells were resuspended in 200 μL PBS, and CD8+ was detected by flow cytometry. + T cell percentage and tumor cells (GFP) + )percentage.

[0049] like Figure 5 The figure shows that CD317 knockdown enhances the anti-tumor effect of CD8+ T cells. As can be seen from the figure, CD317 knockdown and control RKO cells were co-cultured with in vitro expanded T cells at a 1:1 effector-target ratio for 24 hours. All cells were collected, and the ratio of CD8+ T cells (CD8+) to tumor cells (GFP-FITC+) was analyzed by flow cytometry. Figure 5 A). The results showed that the proportion of CD8+ T cells was significantly increased in the CD317 knockdown co-culture system. Figure 5 B), while the proportion of tumor cells decreased ( Figure 5 C) suggests that CD317 knockdown cancer cells are more easily recognized and killed by CD8+ T cells, and CD8+ T cells also proliferate in this process.

[0050] Example 5: Design and validation of CD317-targeting oncolytic virus 1. Design and construction of oncolytic virus vectors targeting CD317 A human CD317-targeting shRNA was constructed using the pDC315 vector. The shRNA was designed, BamHI / HindIII restriction sites were introduced at both ends, and the vector was synthesized. After double digestion with BamHI / HindIII, purification, and ligation, the vector was transformed into JM109 competent cells, and positive clones were obtained by sequencing. The targeting sequence of the CD317 shRNA is 5'-GGGAGAGATCACTACATTAAA-3'.

[0051] 2. CD317 knockdown effect verification 1) Seed 293T cells into 6-well plates, 5 × 10⁶ cells per well. 5 1) Place the cells in a 37℃, 5% CO2 incubator and continue culturing. 2) On the second day, replace the culture medium with fresh medium and add plasmids and transfection reagents according to the table below. Take a 1.5mL centrifuge tube, add 200μL DMEM, mix the plasmids and transfection reagents, let stand at room temperature for 20min, and slowly add to a 6-well plate. Change the medium after 6h.

[0052]

[0053] 3) Collect cells 48 hours after transfection and medium change, extract proteins and perform Western blot analysis.

[0054] 3. Preparation of oncolytic adenovirus The successfully constructed adenovirus shuttle plasmid (pDC315-hTERTp-E1A-EGFP-U6-hBST2) or control plasmid was co-transfected with the packaging plasmid carrying the adenovirus genome (pBHGlox(delta)E1,3Cre) in HEK 293 cells to generate recombinant adenovirus via the Cre / loxP recombinase system.

[0055] like Figure 6 The figure shown is a diagram illustrating the design and validation results of an oncolytic virus targeting CD317. Considering that CD317 has multiple functions including promoting cancer cell proliferation, resisting cell death, and immune escape, this invention further developed an oncolytic virus that targets and knocks down CD317 expression. Figure 6 A), in vitro experiments showed that this virus can efficiently knock down CD317 expression (A), Figure 6 B).

[0056] Example 6: CD317 shRNA oncolytic virus significantly inhibits the progression of RKO xenograft tumors. RKO cells in the logarithmic growth phase were collected by centrifugation, and the cell concentration was adjusted to 3 × 10⁻⁶. 7 Cells / mL. Six- to eight-week-old female NSG mice were randomly divided into two groups of four. Each mouse was subcutaneously injected with 100 μL of cell suspension. When the xenograft reached 200 mm², cells were collected. 3 Treatment began at that time. Each mouse in the OV-shCT and OV-shCD317 groups received an intratumoral injection of 1×10⁻⁶ mmol / L. 9 The corresponding PFU viral solution was injected every two days for a total of four treatments. Every two days, the long and short diameters of the transplanted tumor were measured using calipers to calculate the tumor volume and plot a growth curve. When the tumor volume reached approximately 1500 mm², [further treatment was initiated]. 3 At that time, all mice were euthanized, the tumor tissue was removed and weighed, and the difference in tumor weight between the two groups was compared.

[0057] like Figure 7 The image shows the results of CD317 shRNA oncolytic virus significantly inhibiting the growth of RKO xenograft tumors. Specifically, an RKO mouse xenograft tumor model was constructed, and the tumor volume reached 200 mm². 3 At that time, patients were randomly assigned to two groups and treated with either control oncolytic virus (OV-shCT) or CD317 shRNA oncolytic virus (OV-shCD317) (intratumoral injection, one injection every two days, for a total of four injections). Results showed that CD317 shRNA oncolytic virus significantly delayed the growth of the transplanted tumor. Figure 7At the experimental endpoint, the tumor weight in this group was significantly lower than that in the control oncolytic virus treatment group (A). Figure 7 BC).

Claims

1. A recombinant oncolytic virus, characterized in that, The invention comprises a CD317 inhibitor and an oncolytic virus, wherein the recombinant oncolytic virus is formed by integrating the CD317 inhibitor into the genome of the oncolytic virus; the CD317 inhibitor is a substance capable of inhibiting CD317 gene expression or targeting the degradation of CD317 protein.

2. The recombinant oncolytic virus according to claim 1, characterized in that, The CD317 inhibitor is selected from shRNA or siRNA that targets CD317.

3. The recombinant oncolytic virus according to claim 1, characterized in that, The oncolytic virus is an oncolytic adenovirus, herpes simplex virus, vaccinia virus, Newcastle disease virus, parvovirus, measles virus, or poliovirus, preferably an oncolytic adenovirus.

4. A pharmaceutical composition, characterized in that, It includes the recombinant oncolytic virus as described in any one of claims 1-3 and a pharmaceutically acceptable vector.

5. The use of the recombinant oncolytic virus according to any one of claims 1-3 or the pharmaceutical composition according to claim 4 in the preparation of an antitumor drug.

6. The application according to claim 5, characterized in that, The tumor in question is one that highly expresses CD317.

7. The application according to claim 6, characterized in that, The tumors were selected from liver cancer and breast cancer.

8. The application according to claim 6, characterized in that, The recombinant oncolytic virus or drug composition breaks the immune escape mechanism by reducing CD317 expression in cancer cells, inhibiting cancer cell proliferation, and reducing PD-L1 expression.

9. The application according to claim 5, characterized in that, The anti-tumor drug can be used in combination with T-cell immunotherapy drugs.

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