Synergistic composition for oncolytic virus M1 and application thereof

Abstract

The invention belongs to the technical field of biological medicine, discloses an oncolytic virus M1 synergistic composition and application thereof, and particularly discloses application of low-molecular heparin or pharmaceutically acceptable salt thereof in preparation of a product for enhancing the anti-tumor effect of oncolytic virus. The invention provides a technical scheme for improving the treatment effect of the oncolytic virus M1 in tumor treatment through the low-molecular heparin for the first time, the low-molecular heparin can influence the replication process of the oncolytic virus in cancer cells, the combined application of the low-molecular heparin and the oncolytic virus shows a positive effect, a new direction for optimizing the oncolytic virus therapy is opened up, and the application prospect is broad. And a new possibility is provided for improving the treatment effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an oncolytic virus M1 synergistic composition and an application thereof. Background Art

[0002] Oncolytic viruses (OVs) are a type of virus that can selectively infect and kill tumor cells without affecting normal cells. Due to their unique tumor specificity and immune activation effects, they have received widespread attention in the field of cancer treatment in recent years. Oncolytic virus therapy exerts anti-tumor effects through a variety of mechanisms, including direct lysis of tumor cells, activation of the host's immune response, and induction of changes in the tumor microenvironment. In theory, oncolytic viruses have significant therapeutic potential, especially when they have strong targeting specificity and little damage to healthy tissues, and can provide new treatment options for cancer patients.

[0003] However, although oncolytic virus therapy shows strong clinical application prospects in theory, it still faces a series of challenges in practical applications. First, the ability of oncolytic viruses to replicate in cancer cells is often limited, which directly affects the therapeutic efficacy of the virus. Specifically, after the oncolytic virus enters the host cell, it may be recognized by the host's immune system and trigger an antiviral immune response. The immune system inhibits the further replication and spread of the virus by releasing antiviral cytokines and activating immune cells, which reduces the therapeutic effect of the virus to a certain extent. In addition, the replication of oncolytic viruses inside tumor cells may also encounter multiple resistance mechanisms of tumor cells, including blockade of viral replication pathways, enhanced resistance to apoptosis, and inhibition of viral infection. These factors work together to limit the application of oncolytic virus therapy in clinical treatment.

[0004] In this context, finding effective strategies to enhance the replication ability of oncolytic viruses and improve their efficacy has become one of the research focuses. In recent years, alphavirus M1 has been found to be a highly tumor-specific oncolytic virus with good oncolytic effect 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 Number: CXSL2300588, CXSL2400590, jRCT2063230079). In addition, alphavirus M1 has obtained FDA orphan drug qualification certification in the United States, showing its great potential in cancer treatment.

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

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

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

[0008] The purpose of the third aspect of the present invention is to provide the use of the product of the second aspect of the present invention in the preparation of anti-tumor drugs.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] In order to enhance the replication ability of oncolytic viruses in cancer cells, the applicant's research team conducted in-depth exploration. After systematic exploration and experimental verification, we found that low molecular weight heparin can affect the replication process of oncolytic viruses in cancer cells. The combined use of the two showed positive effects, opened up a new direction for the optimization of oncolytic virus therapy, and provided new possibilities for improving the treatment effect.

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

[0012] (1) Preparing products that enhance the anti-tumor effects of oncolytic viruses;

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

[0014] (3) preparing products that improve the ability of oncolytic viruses to adhere to tumor cells;

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

[0016] (5) preparing a product that increases the number of plaques infected by oncolytic viruses on tumor cells;

[0017] (6) Preparing a product for increasing the amount of oncolytic virus in tumor tissue.

[0018] In some embodiments of the present invention, the tumor comprises at least one of bladder cancer, prostate cancer, brain glioma, melanoma, lung cancer, breast cancer, colorectal cancer and pancreatic cancer; preferably, it is 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 subilin; preferably enoxaparin sodium.

[0021] In some embodiments of the present invention, the oncolytic virus includes at least one of Getavirus, 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 increases the adhesion ability of the oncolytic virus to tumor cells, thereby achieving the purpose of enhancing the anti-tumor effect of the oncolytic virus.

[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 an alkali metal salt or an alkaline earth metal salt.

[0026] In some embodiments of the present invention, the alkali metal salt includes at least one of a sodium salt and a 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 the organic base includes a salt formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0029] In some embodiments of the present invention, the salt formed with an inorganic acid includes a salt formed with the following inorganic acids: at least one of 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 a salt formed with the following organic acids: at least one of 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 a basic amino acid includes a salt formed with the following basic amino acids: at least one of 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 the following acidic amino acids: at least one of aspartic acid and glutamic acid.

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

[0034] The second aspect of the present invention provides a product, which includes 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 the 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 the oncolytic virus in the product is 0.01 to 2 MOI.

[0037] In some embodiments of the present invention, the effective dose of the 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 Subilin; preferably enoxaparin sodium.

[0040] In some embodiments of the present invention, the oncolytic virus includes at least one of Getavirus, 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 drug may also contain pharmaceutically acceptable excipients, such as solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and at least one of carriers.

[0044] In some embodiments of the present invention, the drug may further contain a combined drug, and the drug may be used to treat tumors.

[0045] The third aspect of the present invention provides use of the product of the second aspect of the present invention in the preparation of anti-tumor drugs.

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

[0047] In some embodiments of the present invention, the drug may also contain pharmaceutically acceptable excipients, such as solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and at least one of carriers.

[0048] In some embodiments of the present invention, in order to facilitate medication, the active ingredient can be processed into a specific dosage form with any one or more pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol and microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide and calcium carbonate, etc.), wetting agents (such as water and ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch slurry and syrup, etc.), disintegrants (such as dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants and cross-linked polyvinylpyrrolidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol and micropowder silica gel, etc.), colorants (such as titanium dioxide, sunset yellow, methylene blue and medicinal iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose and povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol and glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid and sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium pyrosulfite and sodium thiosulfate, etc.), antibacterial agents (such as phenol, benzyl alcohol and thimerosal, etc.), and isotonic regulators (such as sodium chloride and glucose, etc.).

[0049] In some embodiments of the present invention, the dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

[0050] In some embodiments of the present invention, the dosage form for administration via the gastrointestinal tract includes at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0051] In some embodiments of the present invention, the dosage forms for administration through the gastrointestinal tract 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, scratched tablets, sustained-release and controlled-release dosage forms such as sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, oral patches, etc.

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

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

[0054] The beneficial effects of the present invention are:

[0055] The present invention proposes for the first time a technical solution for increasing the therapeutic effect of oncolytic virus M1 in treating tumors through 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, opens up a new direction for the optimization of oncolytic virus therapy, and provides new possibilities for improving the therapeutic effect.

[0056] Specifically, experiments have shown that 0.16-2.56 IU / mL of enoxaparin 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Clinical doses of enoxaparin promote the infection and killing of oncolytic virus M1 on various bladder cancer cells; A, C and E are flow cytometry tests for the infection rate of M1 virus in KU-19-19 cells with different concentrations of enoxaparin, with the infection rate of virus alone as the control, A was detected 46 hours after infection, C was detected 44 hours after infection, and E was detected 32 hours after infection; B, D, and F are CCK-8 tests for the cell viability of M1-GFP (MOI shown in the figure) in KU-19-19 cells with different concentrations of enoxaparin, with the virus alone group as the control, B was detected 50 hours after infection, and D was detected at 14 hours after infection. 48 hours after infection, F is detected at 36 hours after infection; G and I are flow cytometry tests for the infection rate of cells with different concentrations of enoxaparin, BFTC-905 (15MOI) (G), UM-UC-3 (10MOI) (I), and the group with virus alone was used as the control; H and J are CCK-8 tests for cell viability, and the group with virus alone was used as the control, BFTC-905 (15MOI) (H), UM-UC-3 (10MOI) (J); p values ​​were determined by one-way ANOVA with Dunnett's test for multiple comparisons and two-way ANOVA with Tukey's multiple comparison test for multiple comparisons. The figure shows the results of three independent repeated experiments. Data are shown as mean ± standard deviation, unmarked means no significant difference, *p<0.05, **p<0.01, ***p<0.001.

[0058] Figure 2Heparin directly binds to M1 virus; A is the superposition of the cryo-EM image of oncolytic virus (gray) and the cryo-EM image of oncolytic virus-heparin (yellow), the larger difference density is marked with blue arrows, and the positions of the five-, three- and two-fold symmetry axes of the icosahedron are marked as pentagons, triangles and ellipses, respectively; B is the ion intensity signal of the mobile phase in the chromatographic column, T = 16 minutes, sample peak; T = 34 minutes, 100mM NaCl; T = 41 minutes, 300mM NaCl; T = 47 minutes, 500mM NaCl; T = 52 minutes, 700mM NaCl; T = 56 minutes, 900mM NaCl; C is the relative content of the virus in the eluted samples collected by qPCR detection, and the data are shown as mean ± standard deviation.

[0059] Figure 3 Enoxaparin promotes the adhesion of oncolytic virus M1 to tumor cells; A is a schematic diagram of the experimental process of the plaque assay; B is the full-well scanning result of the plaque assay, and green is the fluorescence of M1-GFP; C is the statistics of the number of plaques in each well in Figure B; D is the statistics of the average length of plaques in each well in Figure B; E is the result of the virus adhesion experiment; data are shown as mean ± standard deviation, unmarked and ns indicate no significant difference, *p<0.05, **p<0.01, ****p<0.0001.

[0060] Figure 4 Enoxaparin enhances the in vivo therapeutic effect of oncolytic virus M1; A is the plasma concentration measured by an automatic coagulation analyzer 5 hours after subcutaneous injection of different doses of enoxaparin in NCG mice, n=3; B is a schematic diagram of the administration of enoxaparin and oncolytic virus M1 in mice, enoxaparin sodium (ip), oncolytic virus M1 (iv); C~E are individual tumor growth curves (C), tumor growth curves (D) and Kaplan-Meier survival curves (E) of the KU-19-19 model of NCG mice, n=9; p values ​​were compared multiple times at the endpoint (day 14) by one-way analysis of variance with Dunnett's test, and data are shown as mean ± standard deviation, unmarked means no significant difference, *p<0.05, **p<0.01, ***p<0.001.

[0061] Figure 5 Enoxaparin increases the number of viruses in tumor tissues; A is a schematic diagram of the administration and sampling of enoxaparin and oncolytic virus M1 in NCG mice, enoxaparin sodium (ip), oncolytic virus M1 (iv); B is the detection of the number of viruses in various tissues of mice by qPCR; the data were multiple compared by Two-way ANOVA, and the data were displayed as mean ± standard deviation, unmarked means no significant difference, *p<0.05.

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

[0063] The present invention is further described in detail below through specific examples.

[0064] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0065] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0066] Cell lines used in the examples: American Type Culture Collection (UM-UC-3) and Nanjing Foxit Biotechnology Co., Ltd. (KU-19-19, BFTC-905).

[0067] The oncolytic virus M1 with GFP reporter gene (OVM1-GFP virus, denoted as M1-GFP) used in the cell experiment is a virus strain that expresses jellyfish green fluorescent protein (GFP) after genetic modification of the natural alpha virus M1 genome in our laboratory. 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, 1.0 mL: 10000 AXaIU: National Medicine Standard No. HJ20170272.

[0069] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0070] Example

[0071] 1. Experimental Methods

[0072] (1) Determination of the IC 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 inoculated into 48-well plates, with approximately 1×10 cells per well. 4cells, adhered to the wall overnight; take the virus M1 and dilute it in 10 times in equal proportions, so that MOI = 0.1, 1, 10 and set up an uninfected control group. 72 hours after virus infection, add 100μL (5mg / mL) of MTT test solution to each well, incubate in a cell culture incubator for 3-4 hours, then carefully aspirate the supernatant, gently rinse twice with PBS, add 200μL DMSO to each well to dissolve the formazan crystals, and oscillate on a microplate oscillator for 5 minutes to completely dissolve the crystals to form a uniform solution. Detect the absorbance of each well at 570nm on an enzyme reader. Repeat at least 3 times for each cell, and take the average value to draw a dose-effect curve. Relative cell survival rate = (absorbance of drug treatment group-background value) / (absorbance of control group-background value)×100%. The above dose-effect curve IC was calculated using GraphPad Prism 8 software. 50 The MOI value of subsequent experiments was determined.

[0074] (2) Flow cytometry to determine virus infection rate

[0075] Culture KU-19-19 cells (human bladder cancer cells), take cells in the logarithmic growth phase and inoculate them into 48-well plates, with about 1×10 4 cells, adhered overnight; M1-GFP virus was used for infection according to the MOI calculated above, 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, and an uninfected control group was set. 48 hours after virus infection, the cells were digested to prepare a single cell suspension, and the GFP positive rate and GFP average fluorescence intensity were detected by flow cytometry to reflect the virus infection rate and replication. The operation of other bladder cancer cells UM-UC-3 and BFTC-905 was similar, and the concentration of enoxaparin was set to (0.01, 0.04, 0.16, 0.64, 2.56 IU / mL).

[0076] (3) Detection of virus killing ability

[0077] KU-19-19 cells were cultured and cells in the logarithmic growth phase were inoculated into a 48-well plate, with approximately 1×10 4cells, adhered to the wall overnight; M1-GFP virus was infected according to the MOI calculated above, 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, and groups with only different concentrations of enoxaparin and a control group without infection were set up. After 72 hours of virus infection, 10 μL of CCK-8 reaction solution was added to each well, and the absorbance at 450 nm was measured using an ELISA reader after incubation for 1 to 3 hours. Each cell was repeated at least 3 times, and the average value was taken to draw a dose-effect curve. Relative cell survival rate = (absorbance of drug treatment group-background value) / (absorbance of control group-background value)×100%. The operation of other bladder cancer cells UM-UC-3 and BFTC-905 was similar, and the concentration of enoxaparin was changed to (0.01, 0.04, 0.16, 0.64, 2.56 IU / mL).

[0078] (4) Virus plaque assay

[0079] Bladder cancer cells (3×10 3 ) were inoculated in a 96-well plate, and after overnight, M1-GFP (1 MOI) and enoxaparin (0.64 IU / mL) were added. After incubation for 0.5, 1, 2, 4, and 6 h, the supernatant was discarded, washed three times with PBS, and then mixed agarose medium was added. After 48 h of infection, viral infection was observed by an imaging analysis system (Operetta CLS high content analysis system), and the size and number were recorded by taking pictures of the entire well.

[0080] (5) Adhesion test

[0081] KU-19-19 cells were cultured and cells in the logarithmic growth phase were inoculated into 6-well plates, with 5×10 5 Cells were attached overnight. M1-GFP was infected at MOI=50, and the concentration of enoxaparin was (0.16, 0.64 IU / mL), and an uninfected control group was set up. Incubated on ice for 2 hours, washed 3 times with pre-cooled PBS, total RNA was extracted by TRIzol method, and the M1 viral genome content was detected by qRT-PCR.

[0082] (6) Cryo-electron microscopy

[0083] 6000Axa IU enoxaparin and purified oncolytic virus M1 (1×10 9 CCID 50) were incubated overnight at 4°C. Cryo-EM datasets were collected using an elsa cryotransfer stand (Gatan) under a Talos F200C electron microscope (Thermo Fisher). Contrast transfer function (CTF) parameters were estimated using Gctf (real-time ctf determination and correction). Virions were picked and reference-free 2D classification was performed by relion-3. The selected particles were refined by relion-3 and the final reconstruction images were produced. Structures were visualized and compared using UCSF Chimera.

[0084] (7) Affinity chromatography

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

[0086] (8) Detection of HSPG expression on the cell surface

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

[0088] (9) Detection of cell infection rate after antibody blocking HSPG

[0089] Cells in logarithmic growth phase were inoculated into 48-well plates, with approximately 1×10 4 cells, adhered overnight; M1-GFP virus was used for infection at the MOI calculated above, enoxaparin (0.64 IU / mL) and different concentrations of anti-heparan sulfate antibodies (0.5, 2.0, 8.0 μg / mL) were added for co-culture for 48 h, and flow cytometry was used to detect the GFP positivity rate and GFP average fluorescence intensity to reflect the virus infection rate and replication.

[0090] (10) Detection of the number of viruses in various tissues of mouse tumor models

[0091] Human bladder cancer cells KU-19-19 in the logarithmic growth phase were digested into single cell suspensions and injected subcutaneously into NCG mice to establish a model. Mice with uniform tumor formation were randomly divided into 4 groups and given M1 virus (iv) or enoxaparin (ip) + M1 virus in combination. M1 virus was administered intravenously (iv) at 1×10 7 CCID50 / mouse(1×10 7 CCID50 / 0.1mL), enoxaparin subcutaneous injection (ip) loading dose 0.8mg / mouse, maintenance dose 0.4mg / mouse (0.4mg / 0.1mL). Samples were collected on the 2nd, 3rd, and 4th days after administration, and three mice with uniform tumor size and similar weight were selected from each group every day. The mice were euthanized by carbon dioxide inhalation, and the heart, liver, spleen, lung, kidney, brain, and tumor of the mice were collected. Tissue RNA was extracted with TRIzol, and the number of viral copies 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 the FastReal-Time PCR system (Life Technologies) using the Real Time One Step RT-qPCR kit (TIANGEN, FP314). The reaction system and reaction procedures were performed according to the instructions of the kit. The primer 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 Q3S1 fragment was quantified by the standard curve method. All amplification primers and probes were provided by Genscript.

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

[0099] A mouse model was constructed using the same method as (10). 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 (10). The administration lasted for one week. The tumors were measured and the tumor volume (length × width^2 / 2) was calculated every 3 days. The survival of the mice was recorded during the 4-week follow-up and the survival curve was drawn.

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

[0101] A mouse model was constructed using the above method. After tumor formation, the mice were divided into a blank control group, an enoxaparin group, an M1 group, and an enoxaparin + M1 group. The drug administration lasted for one week, and the body weight and food intake of the mice were recorded every three days. Ten days after the drug administration, the ex vivo tissue samples were collected and HE stained to observe tissue pathological changes (heart, liver, spleen, lung, kidney, and brain) to evaluate the safety of the combined therapy.

[0102] 2. Experimental results

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

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

[0105] According to the pharmacokinetic study of enoxaparin in humans, when 1 mg / kg of enoxaparin is injected subcutaneously every 12 hours to treat VTE, the peak and trough values ​​of the patient's steady-state blood drug concentration are 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] In order to explore the mechanism of action of enoxaparin, the present invention uses cryo-electron microscopy (cryo-EM) technology to observe whether enoxaparin can directly interact with oncolytic virus M1. After data collection, the cryo-EM image of oncolytic virus M1 (gray) was superimposed with the image of oncolytic virus M1 after enoxaparin binding (yellow). The results showed that enoxaparin was bound to the surface of the viral protein, indicating that there was a direct interaction between enoxaparin and oncolytic virus M1 ( Figure 2 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 and then eluted stepwise with increasing concentrations of sodium chloride solution ( Figure 2 The eluate was collected and the amount of virus in it was detected by RT-qPCR. The results showed that the M1 virus was only eluted when the sodium chloride concentration reached 300mM, further confirming the direct interaction between the two ( Figure 2 (C) In summary, heparin can directly interact with oncolytic virus M1.

[0108] In order to further explore 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 method to study ( Figure 3 The results showed that enoxaparin significantly increased the number of plaques produced by M1 infection ( Figure 3B and C), but did not change the plaque size ( Figure 3 B and D), suggesting that the synergistic effect of enoxaparin occurs in the early stages of M1 infection. Furthermore, it was observed that enoxaparin increased the adhesion of M1 to KU-19-19 cells ( Figure 3 Middle E), suggesting that enoxaparin exerts its synergistic effect by increasing the adhesion ability of oncolytic virus M1 to cells.

[0109] (3) Clinical doses of enoxaparin selectively increase the amount of intratumoral virus and enhance the tumor-suppressing effect in mice

[0110] In order to ensure that enoxaparin reaches the clinical dose in mice, the present invention first explored the dosage: different doses of enoxaparin were subcutaneously injected and the blood concentration was measured after 5 hours (this is 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 A in the figure). The results showed that when 0.8 mg / mouse enoxaparin was administered, the maximum blood drug concentration could reach about 1 IU / mL, which can simulate the steady-state blood drug concentration in the human body (0.52-1.2 IU / mL). The loading dose is the larger dose at the time of initial administration, usually about twice the maintenance dose, with the purpose of making the blood drug concentration quickly reach or approach the steady-state concentration. Steady-state concentration refers to the stable level reached by the drug in the blood after multiple administrations, at which time the drug input rate is approximately equal to its elimination rate. The peak concentration after the loading dose is usually close to the steady-state concentration, thereby ensuring that the drug can take effect quickly. Therefore, in order to achieve a blood drug concentration of 1 IU / mL to simulate the steady-state blood drug concentration in the human body, the present invention plans to adopt a dosing regimen of 0.8 mg loading dose and 0.4 mg maintenance dose.

[0111] Subsequently, the inventors established a KU-19-19 subcutaneous xenograft tumor model in NCG mice and used Figure 4 The mice were treated with the dosing regimen of medium B. It was observed that enoxaparin significantly enhanced the tumor inhibition effect of M1 and prolonged the survival of mice ( Figure 4C to E in the middle). Furthermore, the inventors used RT-qPCR to detect the number of copies of the M1 viral genome in various organs of mice. The results showed that enoxaparin selectively increased the amount of virus in mouse tumors without changing the amount of virus in other normal organs ( Figure 5 ). Based on the above results, enoxaparin can significantly enhance the in vivo efficacy of oncolytic virus M1.

[0112] (4) The combination therapy of enoxaparin and oncolytic virus M1 has good safety

[0113] Next, in order to evaluate the safety of this combination regimen, the inventors repeated the experiment and recorded in detail the changes in the body weight and food intake of the mice ( Figure 6 The results showed that there were no significant differences in body weight and food intake among the four groups of mice during the first 18 days after tumor implantation. However, on day 21, the average body weight and food intake of mice in the PBS and enoxaparin groups dropped dramatically due to death ( Figure 6 In order to further verify the safety of the combined treatment, the inventors performed pathological HE staining on the main organs of the four groups of mice. The results showed that compared with normal mice, all tumor-bearing mice showed 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 In summary, these experimental results jointly confirmed the safety of this combination therapy.

[0114] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of low molecular weight heparin or a pharmaceutically acceptable salt thereof in any one of (1) to (6): (1) Preparing products that enhance the anti-tumor effects of oncolytic viruses; (2) Improve the adhesion ability of oncolytic viruses to tumor cells; (3) preparing products that improve the ability of oncolytic viruses to adhere to tumor cells; (4) increasing the number of plaques infected by oncolytic viruses on tumor cells; (5) preparing a product that increases the number of plaques infected by oncolytic viruses on tumor cells; (6) Preparing a product for increasing the amount of oncolytic virus in tumor tissue.

2. The use according to claim 1, characterized in that: The tumor includes at least one of bladder cancer, prostate cancer, brain glioma, melanoma, lung cancer, breast cancer, colorectal cancer and pancreatic cancer.

3. The use according to claim 1, characterized in that: The low molecular weight heparin is a heparin fragment with a molecular weight less than 7000Da; Preferably, the low molecular weight heparin includes at least one of enoxaparin sodium, nadroparin sodium, dalteparin sodium and subilin.

4. The use according to any one of claims 1 to 3, characterized in that: The low molecular weight heparin or a pharmaceutically acceptable salt thereof increases the adhesion ability of the oncolytic virus to tumor cells, thereby achieving the purpose of enhancing the anti-tumor effect of the oncolytic virus.

5. The use according to claim 4, characterized in that: The oncolytic virus includes at least one of Getavirus, adenovirus, herpes simplex virus, reovirus, measles virus, Newcastle disease virus, Seneca Valley virus, vesicular stomatitis virus, poliovirus, ECHO enterovirus, coxsackie virus and vaccinia virus, preferably oncolytic virus M1.

6. A product, characterized in that The product comprises low molecular weight heparin or a pharmaceutically acceptable salt thereof and an oncolytic virus.

7. The product according to claim 6, characterized in that The effective dose of the low molecular weight heparin or a pharmaceutically acceptable salt thereof in the product is 0.01 to 2.56 IU / mL; and / or the effective dose of the oncolytic virus in the product is 0.01 to 2 MOI.

8. The product according to claim 7, characterized in that The low molecular weight heparin is a heparin fragment with a molecular weight of less than 7000Da; and / or, the oncolytic virus includes at least one of Geta virus, adenovirus, herpes simplex virus, reovirus, measles virus, Newcastle disease virus, Seneca Valley virus, vesicular stomatitis virus, polio virus, ECHO enterovirus, coxsackie virus and vaccinia virus, preferably oncolytic virus M1.

9. Use of the product according to any one of claims 6 to 8 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor includes at least one of bladder cancer, prostate cancer, brain glioma, melanoma, lung cancer, breast cancer, colorectal cancer and pancreatic cancer.

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