Oncolytic virus m1 potentiating compositions and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses an oncolytic virus M1 synergistic composition and application thereof, and specifically discloses application of low molecular heparin or a pharmaceutically acceptable salt thereof in preparation of a product for enhancing the antitumor effect of an oncolytic virus. The application first proposes a technical scheme for increasing the therapeutic effect of oncolytic virus M1 in tumor treatment by using low molecular heparin, the low molecular heparin can affect the replication process of the oncolytic virus in cancer cells, and the combined use of the two shows a positive effect, opening up a new direction for optimization of oncolytic virus therapy and providing a new possibility for improving the therapeutic effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an oncolytic virus M1 synergistic composition and its application. Background Technology

[0002] Oncolytic viruses (OVs) are a class of viruses that can selectively infect and kill tumor cells without affecting normal cells. Due to their unique tumor specificity and immune-activating effects, they have received widespread attention in the field of cancer treatment in recent years. Oncolytic virus therapy exerts its anti-tumor effects through multiple mechanisms, including directly lysing tumor cells, activating the host's immune response, and inducing changes in the tumor microenvironment. Theoretically, oncolytic viruses have significant therapeutic potential, especially when they exhibit strong targeting specificity and minimal damage to healthy tissues, potentially providing new treatment options for cancer patients.

[0003] However, despite the theoretically promising clinical application of oncolytic virus therapy, a series of challenges remain in practical application. First, the replication capacity of oncolytic viruses within cancer cells is often limited, directly impacting the therapeutic efficacy. Specifically, after entering host cells, oncolytic viruses may be recognized by the host's immune system, triggering an antiviral immune response. The immune system inhibits further viral replication and spread by releasing antiviral cytokines and activating immune cells, which to some extent reduces the therapeutic effect. Furthermore, the replication of oncolytic viruses within tumor cells may encounter multiple resistance mechanisms, including blocking viral replication pathways, enhanced apoptosis resistance, and inhibition of viral infection. These factors combined limit the clinical application of oncolytic virus therapy.

[0004] Against this backdrop, finding effective strategies to enhance the replication capacity of oncolytic viruses and improve their efficacy has become one of the key research focuses. In recent years, alphavirus M1 has been found to be a highly tumor-specific oncolytic virus with good oncolytic effects and immune activation. In 2014, the applicant first identified and reported the oncolytic properties of alphavirus M1 (ProcNatl Acad Sci US A.2014;111(42):E4504-E4512.), and further promoted its related Phase I clinical trials in China, Japan and other places (Clinical Trial Numbers: CXSL2300588, CXSL2400590, jRCT2063230079). In addition, alphavirus M1 has obtained orphan drug designation from the FDA in the United States, showing its great potential in cancer treatment.

[0005] Low molecular weight heparin (LMWH) is a commonly used anticoagulant widely used to prevent thrombosis, treat venous thromboembolism, and myocardial infarction. Compared to conventional heparin, LMWH has better bioavailability and a more stable anticoagulant effect. Among this class of drugs, enoxaparin sodium has become the first-line drug in clinical practice due to its lower bleeding risk and higher bioavailability. Summary of the Invention

[0006] The object of a first aspect of the present invention is to provide the use of low molecular weight heparin or a pharmaceutically acceptable salt thereof.

[0007] The second aspect of the present invention is to provide a product.

[0008] The third aspect of this invention aims to provide the application of the product of the second aspect of this invention in the preparation of antitumor drugs.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] To enhance the replication ability of oncolytic viruses within cancer cells, the applicant's research team conducted in-depth research. Through systematic exploration and experimental verification, we discovered that low molecular weight heparin can affect the replication process of oncolytic viruses within cancer cells. The combined application of the two showed positive effects, opening up a new direction for optimizing oncolytic virus therapy and providing new possibilities for improving treatment outcomes.

[0011] The first aspect of the invention provides the use of low molecular weight heparin or a pharmaceutically acceptable salt thereof in any one of (1) to (6):

[0012] (1) To prepare products that enhance the antitumor effect of oncolytic viruses;

[0013] (2) Enhance the adhesion ability of oncolytic viruses to tumor cells;

[0014] (3) Prepare products that enhance the adhesion ability of oncolytic viruses to tumor cells;

[0015] (4) Increase the number of plaques infected by oncolytic viruses in tumor cells;

[0016] (5) Prepare products that increase the number of plaques in tumor cells infected by oncolytic viruses;

[0017] (6) Prepare products that increase the viral load of oncolytic viruses in tumor tissues.

[0018] In some embodiments of the present invention, the tumor includes at least one of bladder cancer, prostate cancer, glioma, melanoma, lung cancer, breast cancer, colorectal cancer, and pancreatic cancer; preferably bladder cancer.

[0019] In some embodiments of the present invention, the low molecular weight heparin is a heparin fragment with a molecular weight of less than 7000 Da.

[0020] In some embodiments of the present invention, the low molecular weight heparin includes at least one of enoxaparin sodium, nadroparin sodium, dalteparin sodium, and superpirine; preferably enoxaparin sodium.

[0021] In some embodiments of the present invention, the oncolytic virus includes at least one of getta virus, adenovirus, herpes simplex virus, reovirus, measles virus, Newcastle disease virus, Seneca Valley virus, vesicular stomatitis virus, poliovirus, ECHO enterovirus, Coxsackie virus, and vaccinia virus.

[0022] In some embodiments of the present invention, the oncolytic virus is oncolytic virus M1.

[0023] In some embodiments of the present invention, the low molecular weight heparin or a pharmaceutically acceptable salt thereof enhances the antitumor effect of the oncolytic virus by increasing its adhesion to tumor cells.

[0024] In some embodiments of the present invention, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0025] In some embodiments of the present invention, the metal salt includes alkali metal salts and alkaline earth metal salts.

[0026] In some embodiments of the present invention, the alkali metal salt includes at least one of sodium salt and potassium salt.

[0027] In some embodiments of the present invention, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt, and aluminum salt.

[0028] In some embodiments of the present invention, the salt formed with an organic base includes at least one of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0029] In some embodiments of the present invention, the salt formed with the inorganic acid includes at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0030] In some embodiments of the present invention, the salt formed with the organic acid includes at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0031] In some embodiments of the present invention, the salt formed with the basic amino acid includes at least one of the following basic amino acids: arginine, lysine, and ornithine.

[0032] In some embodiments of the present invention, the salt formed with the acidic amino acid includes a salt formed with at least one of the following acidic amino acids: aspartic acid and glutamic acid.

[0033] In some embodiments of the present invention, the product includes reagents, kits, and drugs.

[0034] A second aspect of the invention provides a product comprising low molecular weight heparin or a pharmaceutically acceptable salt thereof and an oncolytic virus.

[0035] In some embodiments of the present invention, the effective dose of low molecular weight heparin or a pharmaceutically acceptable salt thereof in the product is 0.16 to 2.56 IU / mL.

[0036] In some embodiments of the present invention, the effective dose of oncolytic virus in the product is 0.01 to 2 MOI.

[0037] In some embodiments of the present invention, the effective dose of oncolytic virus in the product is 0.01 to 1 MOI.

[0038] In some embodiments of the present invention, the low molecular weight heparin is a heparin fragment with a molecular weight of less than 7000 Da.

[0039] In some embodiments of the present invention, the low molecular weight heparin includes at least one of enoxaparin sodium, nadroparin sodium, dalteparin sodium, and superpirine; preferably enoxaparin sodium.

[0040] In some embodiments of the present invention, the oncolytic virus includes at least one of getta virus, adenovirus, herpes simplex virus, reovirus, measles virus, Newcastle disease virus, Seneca Valley virus, vesicular stomatitis virus, poliovirus, ECHO enterovirus, Coxsackie virus, and vaccinia virus.

[0041] In some embodiments of the present invention, the oncolytic virus is oncolytic virus M1.

[0042] In some embodiments of the present invention, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0043] In some embodiments of the present invention, the medicament may also contain at least one of the following: solvent, propellant, solubilizer, cosolvent, emulsifier, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, flow aid, flavoring agent, preservative, suspending agent, coating material, fragrance, anti-adhesion agent, integrator, penetration enhancer, pH adjuster, buffer, plasticizer, surfactant, foaming agent, defoamer, thickener, encapsulating agent, humectant, absorbent, diluent, flocculant and anti-flocculation agent, filter aid, release inhibitor, and carrier.

[0044] In some embodiments of the present invention, the drug may also contain combination drugs, and the drug may be used to treat tumors.

[0045] A third aspect of the invention provides the use of the product of the second aspect of the invention in the preparation of antitumor drugs.

[0046] In some embodiments of the present invention, the tumor includes at least one of bladder cancer, prostate cancer, glioma, melanoma, lung cancer, breast cancer, colorectal cancer, and pancreatic cancer.

[0047] In some embodiments of the present invention, the medicament may also contain at least one of the following: solvent, propellant, solubilizer, cosolvent, emulsifier, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, flow aid, flavoring agent, preservative, suspending agent, coating material, fragrance, anti-adhesion agent, integrator, penetration enhancer, pH adjuster, buffer, plasticizer, surfactant, foaming agent, defoamer, thickener, encapsulating agent, humectant, absorbent, diluent, flocculant and anti-flocculation agent, filter aid, release inhibitor, and carrier.

[0048] In some embodiments of the present invention, to facilitate administration, the active ingredient may be processed with one or more pharmaceutically acceptable excipients into a specific dosage form. These excipients may be diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose), absorbents (e.g., calcium sulfate, dicalcium phosphate, light magnesium oxide, and calcium carbonate), wetting agents (e.g., water and ethanol), binders (e.g., hydroxypropyl methylcellulose, povidone, starch paste, and syrup), disintegrants (e.g., dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone), and lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and micronized powders). The following are examples of agents: silica gel, colorants (such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, and povidone), solvents (such as water for injection, ethanol, propylene glycol, and glycerin), acid-base adjusters (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate), antioxidants (such as sodium sulfite, sodium metabisulfite, and sodium thiosulfate), antibacterial agents (such as phenol, benzyl alcohol, and thimerosal), and isotonic adjusters (such as sodium chloride and glucose).

[0049] In some embodiments of the present invention, the dosage form of the product includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0050] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0051] In some embodiments of the present invention, the gastrointestinal dosage forms include, but are not limited to, enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratch tablets, sustained-release and controlled-release dosage forms, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, and oral patches.

[0052] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0053] In some embodiments of the present invention, the injectable dosage forms include, but are not limited to, injection solutions, solutions for injection, injection solutions for intravenous infusion, suspensions for injection, sterile powders for injection, intravenous injections, water injections, emulsions for injection, powder injections, injections, sterile powder injections, lyophilized powder injections, etc.

[0054] The beneficial effects of this invention are:

[0055] This invention is the first to propose a technical solution that enhances the therapeutic effect of oncolytic virus M1 in treating tumors by using low molecular weight heparin. Low molecular weight heparin (such as enoxaparin) can affect the replication process of oncolytic virus in cancer cells. The combined application of the two shows positive effects, opening up a new direction for optimizing oncolytic virus therapy and providing new possibilities for improving treatment efficacy.

[0056] Specifically, experiments have shown that enoxaparin at concentrations of 0.16–2.56 IU / mL can significantly promote the infection and killing of oncolytic virus M1, improve the adhesion ability of oncolytic virus M1 to bladder cancer cells, and enhance the therapeutic effect of oncolytic virus M1 on bladder cancer. Attached Figure Description

[0057] Figure 1 This study aimed to promote the infection and killing of oncolytic virus M1 in various bladder cancer cells using clinical doses of enoxaparin. A, C, and E represent the M1 virus infection rate in KU-19-19 cells supplemented with different concentrations of enoxaparin, detected by flow cytometry, with the virus-only infection rate serving as a control. A was detected at 46 hours post-infection, C at 44 hours, and E at 32 hours. B, D, and F represent the cell viability of M1-GFP (MOI shown in the figure) in KU-19-19 cells supplemented with different concentrations of enoxaparin, detected by CCK-8 assay, with the virus-only group serving as a control. B was detected at 50 hours post-infection, and D at [missing data]. At 48 hours post-infection, F represents the result at 36 hours post-infection; G and I represent the cell infection rate detected by flow cytometry with different concentrations of enoxaparin (BFTC-905 (15 MOI))(G) and UM-UC-3 (10 MOI))(I), with the virus-only group serving as the control; H and J represent the cell viability detected by CCK-8 assay, with the virus-only group serving as the control (BFTC-905 (15 MOI))(H) and UM-UC-3 (10 MOI))(J). p-values ​​were determined using one-way ANOVA with Dunnett's test for multiple comparisons and two-way ANOVA with Tukey's test for multiple comparisons. The figures show the results of three independent replicate experiments. Data are presented as mean ± standard deviation; unlabeled data indicate no significant difference, *p<0.05, **p<0.01, ***p<0.001.

[0058] Figure 2This represents the direct binding of heparin to the M1 virus. A shows a superimposed cryo-electron microscopy image of oncolytic virus (gray) and oncolytic virus-heparin (yellow), with larger density differences indicated by blue arrows. The positions of the pentagonal, triangular, and double symmetry axes of the icosahedron are marked as pentagons, triangles, and ellipses, respectively. B shows the ionic intensity signal of the mobile phase in the column: T = 16 min, sample peak; T = 34 min, 100 mM NaCl; T = 41 min, 300 mM NaCl; T = 47 min, 500 mM NaCl; T = 52 min, 700 mM NaCl; T = 56 min, 900 mM NaCl. C shows the relative viral content in the collected eluted samples detected by qPCR, with data presented as mean ± standard deviation.

[0059] Figure 3 To enhance the adhesion of enoxaparin to oncolytic virus M1 to tumor cells; Figure A shows the plaque assay flowchart; Figure B shows the whole-well scan results of the plaque assay, with green representing the fluorescence of M1-GFP; Figure C shows the number of plaques per well in Figure B; Figure D shows the average length of plaques per well in Figure B; Figure E shows the virus adhesion assay results; data are presented as mean ± standard deviation, unlabeled and ns indicate no significant difference, *p<0.05, **p<0.01, ****p<0.0001.

[0060] Figure 4 To enhance the in vivo therapeutic effect of enoxaparin oncolytic virus M1, the study included: A, plasma concentrations measured by an automated coagulation analyzer 5 hours after subcutaneous injection of different doses of enoxaparin in NCG mice (n=3); B, a schematic diagram of enoxaparin and oncolytic virus M1 administration in mice (enoxaparin sodium (ip), oncolytic virus M1 (iv); C–E, individual tumor growth curves (C), tumor growth curves (D), and Kaplan-Meier survival curves (E) in the NCG mouse KU-19-19 model (n=9); p-values ​​were determined by one-way ANOVA and Dunnett's test for multiple comparisons at the endpoint (day 14). Data are presented as mean ± standard deviation. Unlabeled data indicate no significant difference. *p<0.05, **p<0.01, ***p<0.001.

[0061] Figure 5 Enoxaparin was used to increase the viral load in tumor tissues. A shows a schematic diagram of enoxaparin and oncolytic virus M1 administration and sampling in NCG mice (enoxaparin sodium, ip; oncolytic virus M1, iv). B shows the viral load in various mouse tissues detected by qPCR. Data were analyzed using two-way ANOVA for multiple comparisons. Data are presented as mean ± standard deviation. Unlabeled data indicate no significant difference. *p < 0.05.

[0062] Figure 6To assess the safety of the combination therapy; where A is a schematic diagram of enoxaparin and oncolytic virus M1 administration sampling in NCG mice, enoxaparin sodium (ip), oncolytic virus M1 (iv); B-C are the food intake (B) and average body weight change curves (C) of the subcutaneous tumor-bearing mice constructed in Figure A, n=5; D is the HE-stained histopathological image of tumor-bearing mice after ten days of combination treatment, scale bar 100μm. Detailed Implementation

[0063] The present invention will be further described in detail below through specific embodiments.

[0064] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0065] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0066] The cell lines used in the examples are: American Center for Type Culture Collection (UM-UC-3) and Nanjing Fuxai Biotechnology Co., Ltd. (KU-19-19, BFTC-905).

[0067] The oncolytic virus M1 (OVM1-GFP virus, denoted as M1-GFP) carrying the GFP reporter gene used in cell experiments is a viral strain in our laboratory that expresses jellyfish green fluorescent protein (GFP) after genetic modification of the genome of the natural alpha virus M1. The OVM1 used in animal experiments was provided by Guangzhou Weirongte Pharmaceutical Technology Co., Ltd.

[0068] The enoxaparin used in the examples was purchased from Sanofi Ltd., 1.0 mL: 10000 A Xa IU: National Drug Approval Number HJ20170272.

[0069] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0070] Example

[0071] 1. Experimental Methods

[0072] (1) Determine the IC50 of the virus 50 value

[0073] Bladder cancer cells (KU-19-19, BFTC-905, UMUC-3 cells) were cultured, and cells in the logarithmic growth phase were seeded into 48-well plates, approximately 1 × 10⁶ cells per well. 4Cells were incubated overnight. M1 alphavirus was serially diluted 10-fold to achieve MOIs of 0.1, 1, and 10, with a control group not infected. 72 hours after infection, 100 μL (5 mg / mL) of MTT solution was added to each well, and the cells were incubated for 3–4 hours. The supernatant was then carefully aspirated, and the cells were gently washed twice with PBS. 200 μL of DMSO was added to each well to dissolve the formazan crystals, and the cells were shaken on a microplate shaker for 5 minutes to completely dissolve the crystals and form a homogeneous solution. The absorbance at 570 nm was measured using a microplate reader. Each cell line was repeated at least three times, and the average value was used to plot a dose-response curve. Relative cell viability was calculated as (absorbance of the drug-treated group - background value) / (absorbance of the control group - background value) × 100%. The IC50 of the dose-response curve was calculated using GraphPad Prism 8 software. 50 The value is used to determine the MOI value for subsequent experiments.

[0074] (2) Flow cytometry determination of viral infection rate

[0075] KU-19-19 cells (human bladder cancer cells) were cultured, and cells in the logarithmic growth phase were seeded into 48-well plates, approximately 1 × 10⁶ cells per well. 4 Cells were cultured overnight and adhered to the culture vessel. M1-GFP virus was used to infect cells at the calculated MOI, and different concentrations of enoxaparin (0.01, 0.04, 0.16, 0.64, 2.56, 10.24, 41, 164 IU / mL) were added for co-culture. A control group without infection was also included. 48 hours after virus infection, cells were digested to prepare single-cell suspensions. Flow cytometry was used to detect the GFP positivity rate and average GFP fluorescence intensity to reflect the virus infection rate and replication status. The procedure was similar for other bladder cancer cells UM-UC-3 and BFTC-905, with enoxaparin concentrations set at (0.01, 0.04, 0.16, 0.64, 2.56 IU / mL).

[0076] (3) Detecting the virus's killing ability

[0077] KU-19-19 cells were cultured, and cells in the logarithmic growth phase were seeded into 48-well plates, approximately 1 × 10⁶ cells per well. 4Cells were incubated overnight and adhered to the culture vessel. M1-GFP virus was used to infect cells at the calculated MOI, and different concentrations of enoxaparin (0.01, 0.04, 0.16, 0.64, 2.56, 10.24, 41, 164 IU / mL) were added for co-culture. Groups with only different concentrations of enoxaparin and a control group without infection were also established. 72 h after virus infection, 10 μL of CCK-8 reaction solution was added to each well, and after incubation for 1–3 h, the absorbance at 450 nm was measured using a microplate reader. Each cell was repeated at least three times, and the average value was used to plot a dose-response curve. Relative cell viability = (absorbance of drug-treated group - background value) / (absorbance of control group - background value) × 100%. The procedure was similar for other bladder cancer cells UM-UC-3 and BFTC-905, except the enoxaparin concentration was changed to (0.01, 0.04, 0.16, 0.64, 2.56 IU / mL).

[0078] (4) Viral plaque experiment

[0079] Bladder cancer cells (3×10) 3 Inoculate into 96-well plates and incubate overnight. Then add M1-GFP (1 MOI) and enoxaparin (0.64 IU / mL). After incubation for 0.5, 1, 2, 4, and 6 h, discard the supernatant, wash three times with PBS, and then add mixed agarose medium. 48 h after infection, observe viral infection using an imaging analysis system (Operetta CLS high-content analysis system), and record the size and number of cells by photographing the entire well.

[0080] (5) Adhesion test

[0081] KU-19-19 cells were cultured, and cells in the logarithmic growth phase were seeded into 6-well plates at a density of 5 × 10⁶ cells per plate. 5 Cells were incubated overnight. M1-GFP cells were infected at an MOI of 50 with enoxaparin concentrations of 0.16 and 0.64 IU / mL, and a control group was included without infection. Cells were incubated on ice for 2 hours, washed three times with pre-cooled PBS, and total RNA was extracted using the TRIzol method. M1 viral genome content was detected by qRT-PCR.

[0082] (6) Cryo-electron microscopy

[0083] 6000 Axa IU enoxaparin and purified oncolytic virus M1 (1×10⁻⁶) 9 CCID 50Incubated overnight at 4°C. Cryo-electron microscopy datasets were collected using an Elsa cryotransfer scaffold (Gatan) under a Talos F200C electron microscope (Thermo Fisher). Contrast transfer function (CTF) parameters were estimated using Gctf (real-time CTF determination and correction). Viral particles were selected and classified in 2D without reference using relion-3. relion-3 refined the selected particles, producing the final reconstructed images. The structures were visualized and compared using UCSF chimeras.

[0084] (7) Affinity chromatography

[0085] M1-GFP virus purified in dPBS was applied to a 1 mL HiTrap heparin-agarose gel column (GE Healthcare, 17040601). The column was washed with 10 mL dPBS, and bound proteins were eluted with a NaCl gradient of 100, 300, 500, 700, and 900 mM. 100 μL of the NaCl eluent at different concentrations was collected for RNA extraction and qRT-PCR detection.

[0086] (8) Detection of heparin sulfate proteoglycan (HSPG) expression on cell surface

[0087] Take 1×10 cells in the logarithmic growth phase 5 Cells were digested into single-cell suspensions, balanced, and centrifuged. The supernatant was discarded, and 0.1 mL of PBS buffer and 1 μL of anti-heparan sulfate antibody were added. The mixture was incubated at 4°C in the dark for 30 min. 1 mL of PBS buffer was added to each tube, vortexed, and centrifuged. The supernatant was discarded. This step was repeated twice. 0.1 mL of PBS buffer and 1 μL of fluorescent secondary antibody (APC) were added, mixed, and incubated at 4°C in the dark for 30 min. 1 mL of PBS buffer was added to each tube, vortexed, and centrifuged. The supernatant was discarded. This step was repeated twice. The stained cells were resuspended in 1 mL of PBS buffer, vortexed, and the fluorescence intensity was detected using a flow cytometer selected from the appropriate detection channels.

[0088] (9) Detect the infection rate of cells after antibody blocking of HSPG

[0089] Logarithmic growth phase cells were seeded into 48-well plates, approximately 1 × 10⁶ cells per well. 4 Cells were incubated overnight and adhered to the culture vessel. M1-GFP virus was used to infect cells at the calculated MOI, and enoxaparin (0.64 IU / mL) and different concentrations of anti-heparan sulfate antibody (0.5, 2.0, 8.0 μg / mL) were added and co-cultured for 48 h. Flow cytometry was used to detect the GFP positivity rate and the average GFP fluorescence intensity to reflect the viral infection rate and replication status.

[0090] (10) Detection of viral load in various tissues of mouse tumor model

[0091] Human bladder cancer cells (KU-19-19) in the logarithmic growth phase were digested into a single-cell suspension and injected subcutaneously into NCG mice to establish a tumor model. Mice with uniform tumor formation were randomly divided into four groups, receiving either M1 virus (iv), enoxaparin (ip), or a combination of M1 virus and enoxaparin, respectively. Specifically, M1 virus was administered intravenously at a dose of 1 × 10⁻⁶ cells / day (iv). 7 CCID50 / mouse(1×10 7 Enoxaparin was administered subcutaneously (ip) at a loading dose of 0.8 mg / mouse and a maintenance dose of 0.4 mg / mouse (0.4 mg / 0.1 mL). Samples were collected on days 2, 3, and 4 post-administration. Three mice with uniform tumor size and similar body weight were selected from each group daily. Mice were euthanized using the carbon dioxide inhalation method. Heart, liver, spleen, lung, kidney, brain, and tumor tissues were collected. RNA was extracted from the tissues using TRIzol, and viral copy number was detected by qPCR.

[0092] (11) qPCR detection of viral copy number

[0093] Total RNA was extracted using TRIzol reagent (Life Technologies). Quantitative PCR experiments were performed on a FastReal-Time PCR system (Life Technologies) using the Real-Time One Step RT-qPCR kit (TIANGEN, FP314). The reaction system and procedure followed the kit's instructions. The primer and probe sequences used are as follows:

[0094] Q3S1-F: 5'-GGGATTCACTACACCTGCTTAGAC-3' (SEQ ID NO: 1);

[0095] Q3S1-R: 5'-GCTGACTCTGTCTGCGTAACC-3' (SEQ ID NO: 2);

[0096] Q3S1-Probe: 5'-CTCTCATCAGCAGCGAGCCTCCT-3' (SEQ ID NO: 3);

[0097] The copy number of the Q3S1 fragment was quantitatively calculated using the standard curve method. All amplification primers and probes were provided by Genscript.

[0098] (12) Verify the effectiveness of combination therapy

[0099] The same method as in (10) was used to construct a mouse model. After tumor formation, the mice were divided into a blank control group, an enoxaparin group, an M1 group, and an enoxaparin + M1 group. The administration method and dosage were the same as in (10). The administration time was one week. The tumor volume (length × width 2 / 2) was measured every 3 days. The survival status of the mice was recorded and a survival curve was plotted after 4 weeks of follow-up.

[0100] (13) Verify the safety of combination therapy

[0101] Mouse models were constructed using the same method described above. After tumor formation, the mice were divided into a blank control group, an enoxaparin group, an M1 group, and an enoxaparin + M1 group. The administration period was one week. The weight and food intake of the mice were recorded every three days. Ten days after administration, the mice were harvested and the ex vivo tissue samples were stained with hematoxylin and eosin (HE) to observe histopathological changes (heart, liver, spleen, lung, kidney, and brain) in order to evaluate the safety of the combination therapy.

[0102] 2. Experimental Results

[0103] (1) Clinical doses of enoxaparin promote the infection and killing of various tumor cells by oncolytic virus M1.

[0104] This invention treated the human bladder cancer cell line KU-19-19 with different concentrations (0.01–164 IU / mL) of enoxaparin, and then infected these cells with an oncolytic virus M1 carrying the GFP reporter gene (hereinafter abbreviated as M1-GFP) at an infection dose of MOI = 0.1. The results showed that, within a concentration range of 2.56–164 IU / mL, enoxaparin reduced the infection rate of M1 in a dose-dependent manner. Figure 1 (A) This antiviral effect is consistent with results observed on most other viruses. However, at lower concentrations (0.01–0.64 IU / mL), the inventors unexpectedly observed that enoxaparin dose-dependently promoted infection and killing of M1. Figure 1 (A and B). To verify this phenomenon, the inventors repeated the above experiments under different infection conditions (MOI = 0.5 and 1) and on more bladder cancer cell lines (BFTC-905 and UM-UC-3), all of which demonstrated that low-dose enoxaparin significantly promoted the infection and killing of oncolytic virus M1. Figure 1 (C-J).

[0105] According to pharmacokinetic studies of enoxaparin in humans, when enoxaparin was administered subcutaneously at a dose of 1 mg / kg every 12 hours for VTE treatment, the peak and trough steady-state plasma concentrations were approximately 1.2 and 0.52 IU / mL, respectively (Bruno R, Baille P, Retout S, et al. Population pharmacokinetics and pharmacodynamics of enoxaparin unstable angina and non-ST-segment elevation myocardial infarction. British Journal of Clinical Pharmacology. 2003; 56(4):407-414. doi:10.1046 / j.1365-2125.2003.01904.x). Within this concentration range, enoxaparin has a promoting effect on oncolytic virus M1. The above results suggest that clinical doses (0.01–0.64 IU / mL) of enoxaparin can enhance the therapeutic effect of oncolytic virus M1 on bladder cancer.

[0106] (2) Enoxaparin directly binds to M1 and promotes the adhesion of M1 to tumor cells.

[0107] To investigate the mechanism of action of enoxaparin, this invention employed cryo-electron microscopy (cryo-EM) to observe whether enoxaparin could directly interact with oncolytic virus M1. After data collection, cryo-EM images of oncolytic virus M1 (gray) were overlaid with images of oncolytic virus M1 after enoxaparin binding (yellow). The results showed that enoxaparin bound to the surface of the viral protein, indicating a direct interaction between enoxaparin and oncolytic virus M1. Figure 2 (A). Next, heparin-coupled affinity chromatography was used to further investigate whether heparin could directly bind to the M1 virus. First, the M1 virus was loaded onto a heparin column, followed by stepwise elution with increasing concentrations of sodium chloride solution (A). Figure 2 (B) The eluent was collected and the viral load was detected by RT-qPCR. The results showed that M1 virus was eluted only when the sodium chloride concentration reached 300 mM, further confirming the direct interaction between the two. Figure 2 (C) In summary, heparin can directly interact with oncolytic virus M1.

[0108] To further investigate whether the synergistic effect of enoxaparin on oncolytic virus M1 infection occurs in the early or late stages of infection, the inventors used the viral plaque assay for research. Figure 3 (A). The results showed that enoxaparin significantly increased the number of plaques produced by M1 infection ( Figure 3(B and C), but without changing the plaque size ( Figure 3 (Figures B and D) suggest that the synergistic effect of enoxaparin occurs in the early stages of M1 infection. Furthermore, enoxaparin was observed to enhance the adhesion of M1 cells to KU-19-19 cells. Figure 3 The results suggest that enoxaparin exerts its synergistic effect by increasing the adhesion of oncolytic virus M1 to cells.

[0109] (3) Clinical doses of enoxaparin selectively increased intratumoral viral load and enhanced tumor-suppressive effects in mice.

[0110] To ensure that enoxaparin reaches the clinical dose in mice, this invention first determined the dosage: different doses of enoxaparin were injected subcutaneously and the blood concentration was measured after 5 hours (this was the peak blood concentration). (Febbraro S, Leal Martínez-Bujanda J, Nieto Magro C, et al. Bioavailability study of Enoxaparin Sodium Chemi (80 mg / 0.8 mL) and Clexane (80 mg / 0.8 mL) subcutaneous injection in healthy adults[J]. International Journal of Clinical Pharmacology and Therapeutics, 2021, 59(11):734-744.) Figure 4 (See Figure A). Results showed that when 0.8 mg / mouse enoxaparin was administered, the maximum plasma concentration reached approximately 1 IU / mL, which can simulate the steady-state plasma concentration in humans (0.52–1.2 IU / mL). The loading dose is the largest initial dose, typically about twice the maintenance dose, designed to rapidly reach or approach the steady-state plasma concentration. Steady-state concentration refers to the stable level of a drug in the blood after multiple administrations, at which point the rate of drug infusion is approximately equal to its rate of elimination. The peak concentration after the loading dose is usually close to the steady-state concentration, thus ensuring the drug can exert its effect rapidly. Therefore, to achieve a plasma concentration of 1 IU / mL to simulate the steady-state plasma concentration in humans, this invention plans to use a dosing regimen of a 0.8 mg loading dose and a 0.4 mg maintenance dose.

[0111] Subsequently, the inventors constructed a KU-19-19 subcutaneous xenograft model in NCG mice and used methods such as... Figure 4 Treatment with the B-type drug regimen showed that enoxaparin significantly enhanced the antitumor effect of M1 and prolonged the survival of mice. Figure 4(C-E). Furthermore, the inventors used RT-qPCR to detect the copy number of the M1 virus genome in various mouse organs. The results showed that enoxaparin selectively increased the viral load in mouse tumors without altering the viral load in other normal organs. Figure 5 In summary, enoxaparin significantly enhances the in vivo efficacy of oncolytic virus M1.

[0112] (4) Enoxaparin combined with oncolytic virus M1 therapy has a good safety profile.

[0113] Next, to assess the safety of this combination therapy, the inventors repeated the experiment and meticulously recorded changes in mouse body weight and food intake. Figure 6 (A). The results showed that there were no significant differences in body weight and food intake among the four groups of mice in the first 18 days after tumor implantation. However, on day 21, due to death, the average body weight and food intake of mice in the PBS group and enoxaparin group decreased sharply. Figure 6 (B-C). To further verify the safety of the combined treatment, the inventors performed pathological HE staining on the major organs of the four groups of mice. The results showed that, compared with normal mice, all tumor-bearing mice exhibited varying degrees of pathological changes in the liver, spleen, and lungs, but no significant difference was found between the combined treatment group and the PBS group. Figure 6 (D). In summary, these experimental results collectively confirm the safety of this combination therapy.

[0114] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of low molecular weight heparin in the preparation of products that enhance the antitumor effects of oncolytic viruses: The low molecular weight heparin is enoxaparin sodium; The oncolytic virus is oncolytic virus M1; The tumor is bladder cancer.

2. The application according to claim 1, characterized in that, The low molecular weight heparin enhances the antitumor effect of oncolytic viruses by increasing their adhesion to tumor cells, increasing the number of plaques infected by oncolytic viruses on tumor cells, and / or increasing the viral load of oncolytic viruses in tumor tissue.

3. The use of a product in the preparation of an antitumor drug, said product comprising enoxaparin sodium and oncolytic virus M1, wherein the tumor is bladder cancer.

4. The application according to claim 3, characterized in that, The effective dose of enoxaparin sodium in the product is 0.01–2.56 IU / mL; and / or, the effective dose of oncolytic virus M1 in the product is 0.01–2 MOI.

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