Preparation and application of intravenous injection oncolytic virus capable of strengthening radiotherapy curative effect

Abstract

The invention discloses preparation and application of an intravenous injection oncolytic virus capable of strengthening a radiotherapy curative effect, and belongs to the field of nano-drugs. According to the invention, PEI-Se-Se-PEG is utilized to modify the surface of an oncolytic virus with negative charges, so that an invisible oncolytic virus suitable for intravenous injection, which is called AD (at) PSSP for short, is developed. Compared with the prior art, through intravenous injection of the AD (at) PSSP, the circulation time of the oncolytic virus in blood can be remarkably prolonged, and the safety is improved; in addition, not only is the inhibition effect of radiotherapy on whole-body tumor growth effectively improved, but also lasting immune memory can be activated, and tumor recurrence can be inhibited.

Description

Technical Field

[0001] The present invention relates to the field of nanomedicine, and more particularly to the preparation and application of an intravenous oncolytic virus capable of enhancing the therapeutic effect of radiotherapy. Background Art

[0002] Oncolytic viruses (OVs) represent an emerging immunotherapy approach. In recent years, significant progress has been made in clinical trials for diseases such as melanoma and glioma. However, for patients with diffuse or recurrent tumors, radiotherapy (RT) is often required to improve the therapeutic effect. Despite this, we carefully reviewed these related clinical trials and found some urgent problems to be solved. First, the antigen utilization rate of OVs is low, and a large amount of antigens will be produced after tumor RT. In addition, the clinical application of OVs combined with RT is mainly limited to intratumoral injection, which greatly limits their therapeutic effect on deep and metastatic lesions. Second, although intravenous infusion of OVs seems to be a better method for treating metastatic tumors and tumors of deep organ origin, in clinical practice, the efficacy of intravenous administration is far from expected. Once OVs enter the blood circulation, the heterogeneity of their surface antigen proteins can activate the body's own immune system, causing antibodies and complement in the peripheral blood to bind to the virus to form immune complexes, and prompting the virus to be internalized and cleared by immune organs such as the liver and spleen, thereby greatly reducing the circulation time of OVs in the peripheral blood. Therefore, there is an urgent need to develop more routes of administration to expand the scope of application of OVs therapy. At the same time, we also need to work hard to find ways to amplify the ability of OVs combined with RT to clear tumor cells, such as enhancing the infection ability of OVs to enhance the DNA damage effect and the ability of specific immune clearance of tumors. These are challenges we need to overcome.

[0003] Chemical modification and biofilm encapsulation have been shown to significantly optimize the pharmacokinetic properties of therapeutic drugs in vivo. Chemical modification of gene delivery vectors based on cationic compounds can improve the uptake rate of target cells, among which polyethyleneimine (PEI) is known for its excellent nucleic acid aggregation and stabilization ability. In the field of viral gene delivery, PEI is often regarded as the "gold standard" due to its superior transgene expression induction and has been shown to be more effective than other commonly used cationic polymers such as chitosan and polyamide. Complexes of PEI and nucleic acids can be reproducibly prepared by simple mixing. In addition, PEI multiplexes are stable at room temperature, allowing long-term storage of their frozen complexes while maintaining sufficient activity. PEI exists in two different chemical structural forms, namely branched and linear PEI. Under low molecular weight conditions, PEI is often cross-linked with biodegradable linkers to maintain excellent nucleic acid aggregation while improving biocompatibility. Branched PEI is synthesized by ring-opening polymerization of aziridine, while linear PEI is obtained by post-modification of other polymers such as poly(2-oxazoline)). Both synthetic methods have been described in detail and provide efficient and cost-effective routes for large-scale production. PEI vectors have multifunctional properties due to the widespread presence of amine groups, which can meet various gene delivery requirements.

[0004] Adenovirus invasion of host cells depends on the interaction between receptors and ligands, and cells with low expression of coxsackie and adenovirus receptor (CAR) have poor infection efficiency of AD virus. In order to improve the infection efficiency of AD virus, researchers have developed an AD complex (AD / DA3) coated with bile acid conjugated PEI. AD / DA3 significantly improved the infection efficiency in cancer cells with moderate CAR expression or CAR negative. Compared with naked AD, RdB-KOX / DA3 significantly enhanced cell apoptosis, reduced angiogenesis, reduced cell proliferation, and enhanced the replication of active viruses in human tumor xenograft models. These results indicate that PEI-modified AD virus can improve the efficacy of oncolytic AD virus therapy, especially in CAR-restricted tumors.

[0005] Since PEI is positively charged and tumor antigens are usually negatively charged, PEI has the potential to capture negatively charged antigens. Similar to the aforementioned AD-Mal, AD-PEI that fails to enter tumor cells in time also has the potential to drain captured antigens to lymph nodes and stimulate specific immune killing effects. However, compared with Mal, the stability and efficiency of binding to antigens need to be further investigated. In addition, it is important that PEI can also be used as a carrier for delivering antigens, enhancing innate and adaptive immune responses, and showing the characteristics of an immune adjuvant. Protease-containing endosomes are responsible for intracellular sorting, and endosomal escape of vaccine complexes is crucial for effective antigen release. Endolysosomal degradation may hinder effective release. The pH value in the endosome will drop from the physiological pH value of 7.4 in the early endosome to the pH value of 5.0 in the lysosome due to the action of the membrane-bound ATPase pump. PEI exhibits high buffering capacity at almost all pH values ​​and is therefore a compound with high endosomal escape activity. After endosome acidification, cationic PEI can cause endosome rupture through the "proton sponge" effect; protonation causes passive influx of chloride ions, followed by influx of water, which eventually leads to osmotic swelling and endosome rupture, allowing PEI-based vaccines to escape into the vesicle. After the antigens in PEI-based vaccines escape from the endosomes, they stimulate specific immune responses of T cells through a cross-presentation mechanism. In addition, PEI-based vaccines have immunoreactivity that promotes systemic immune responses. One study found that PEI can activate CD8 and CD4 T cells in mixed Th1 / Th2 immune responses through in vivo immunization studies. However, the specific mechanism of action of PEI immunization is still unclear. The immune activity of PEI may be attributed to the activation of multiple molecular pathways, such as the TLR pathway, the pyrin domain-containing NLRP3 inflammasome pathway, and the danger signaling pathway. Therefore, OVs modified with PEI (OVs-PEI) are expected to enhance DNA damage after RT by enhancing viral infectivity, and OVs-PEI can also enhance the immune killing effect after RT by capturing and presenting antigens.

[0006] However, the large amount of positive charge carried by PEI is not conducive to intravenous injection. In addition, the use of polymer methods such as PEG has significantly increased the blood circulation half-life of drugs and improved their biocompatibility. Moreover, encapsulating drugs in biofilms can optimize their distribution in the body and achieve homologous targeting by leveraging the natural homing properties of the body. Therefore, PEGylation or biofilm modification of OVs-PEI can promote the intravenous application of the complex. However, as a biologically active entity, OVs may suffer significant loss of infectious properties when chemically modified or encapsulated in biofilms. Therefore, when administering OVs-PEI intravenously, the biggest challenge is to develop strategies that can remove the protective layer after reaching the tumor microenvironment to restore the original infectious properties of the virus.

[0007] The controlled release of nanomedicines, by virtue of their responsiveness, can not only ensure the precise delivery of targeted drugs, but also precisely deconstruct the protective nanoshell by rearranging the molecular structure upon receiving the activation signal. Such responsive systems are designed to exploit the specific physicochemical properties inherent to the tumor microenvironment, such as enhanced permeability, acidic pH, or overexpressed enzymes. On the exogenous side, the introduction of external excitation factors such as magnetic fields, focused ultrasound, or RT further expands the strategic repertoire of nanomedicines in terms of precise navigation and drug release. Of particular note, the synthesis of reactive oxygen species (ROS)-responsive polymer carriers and their medical applications have stimulated extensive research interest. A number of ROS-responsive polymers for drug delivery systems have been developed and have shown advantages over traditional drug delivery methods in terms of therapeutic effects. At present, many different types of ROS-responsive polymer carriers have been explored, including thioether-containing polymers, selenium (Se) / tellurium-containing polymers, arylboronic acid / ester-containing polymers, aryl oxalates, and polyproline. These ROS-responsive polymers usually cause chemical bond breakage and / or phase transition from hydrophobic to hydrophilic phase, which helps release the drug in the carrier. Of particular interest are Se-containing polymers, which are an element that is essential to the human body and plays a key role in protecting cells from oxidative damage. Se deficiency in the human body may lead to Keshan disease and depression. Due to the higher atomic number and lower electronegativity of Se, the chemical bond energy of Se-containing polymers is lower than that of sulfur-containing polymers (C-Se bond 244 kJ / mol, Se-Se bond 172 kJ / mol, CS bond 272 kJ / mol, SS bond 240 kJ / mol). Se-containing polymers are more sensitive to ROS, and they can be oxidized to Se and selenone, which changes from hydrophobic to hydrophilic through a mechanism similar to thioethers. After the diselenide bond is oxidized to selenic acid, it can be reduced to selenol and finally cleaved, similar to the reaction mechanism of disulfide bonds. Compared with sulfur-containing polymers, Se-containing polymers are more sensitive to oxidants and are suitable for environments with low ROS concentrations. For a long time, interest in the synthesis of Se-containing polymers has been limited because Se-containing compounds and diselenide bonds are unstable in the presence of oxygen and the polymers have poor solubility. However, a pioneering study by Xu and Zhang et al. in 2010 showed that the solubility of diols could be improved by introducing diselenide groups into their structure. They synthesized amphiphilic diselenide polyurethanes by stepwise polymerization from toluene diisocyanate and diselenide-containing diols. Since then, many Se-containing copolymers have been developed, leading to the synthesis of a large number of ROS-responsive polymer carriers. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention provides a preparation and application of intravenous OVs that can enhance the efficacy of RT. The present invention develops a "stealth" OVs (AD@PEI-SeSe-PEG, AD@PSSP for short) suitable for intravenous injection to cooperate with RT. PEI-SeSe-PEG modifies negatively charged OVs to resist neutralizing antibodies and prolong blood circulation. When circulating around the tumor and encountering ROS generated after RT, the Se-Se bond in AD@PSSP breaks, PEG falls off, and the infection ability of OVs is restored. In addition, dePEGylated PEI may enhance the ability of OVs to infect tumor cells through electrostatic interactions. In addition, we observed that OVs can attenuate the activation of the CHEK1-CDK pathway, thereby enhancing DNA damage after radiotherapy. Finally, AD-PEI is used as an in situ tumor vaccine carrier to capture tumor antigens after RT and deliver them to dendritic cells, generate antigen-specific immune responses, effectively ablate distant tumors, and be accompanied by lasting immune memory.

[0009] The OVs modification strategy described in the present invention is expected to lay the foundation for clinical trials involving OVs combined with RT and subsequent clinical deployment. In addition, the OVs also have the characteristics of good biocompatibility, strong stability, high safety, and high bioavailability.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] The present invention provides an intravenous oncolytic virus (OVs)-AD@PSSP that can enhance the therapeutic effect of RT.

[0012] The oncolytic virus is preferably a genetically engineered oncolytic adenovirus AD expressing the telomerase (TERT) promoter and AD5 enhancer. 11 .

[0013] The oncolytic virus modification group is polyethyleneimine-diselenide-polyethylene glycol (PEI-SeSe-PEG, PSSP)

[0014] The present invention provides a method for preparing the AD@PSSP, comprising:

[0015] OVs were incubated with excess PEI-SeSe-PEG at room temperature, and then the unreacted active groups were removed through a filter membrane and an ultrafiltration tube. PBS was added to the ultrafiltration tube for resuspending to obtain AD@PSSP.

[0016] Preferably, the preparation method of AD@PSSP is as follows: PEI-SeSe-PEG solution is mixed with AD, and then incubated at room temperature to promote full binding between the two. Then, the unreacted active groups are removed by a filter membrane and an ultrafiltration tube, and PEG is stained with barium iodide to determine the maximum reaction dosage (PEG bands are not significantly widened or deepened).

[0017] Among them, according to the ratio of solute number to virus particle number, AD 11 :PEI-SeSe-PEG(1.6×10 6 / vp).

[0018] Preferably, PEI-SeSe-PEG, the molecular weight of PEG is 20KDa. Our preliminary experiments and related literature reported that the resistance of PEGylated AD to neutralizing antibodies and macrophage phagocytosis increases with the increase of PEG molecular weight, reaching the highest at 20kDa.

[0019] Preferably, in the ultracentrifuge tube, the low speed centrifugation speed used is 5000-10000 rpm, and the centrifugation is performed for 5-10 min;

[0020] Preferably, the pore size of the filter membrane used is 0.45 μm and 0.22 μm;

[0021] Preferably, the molecular weight cut-off of the ultrafiltration tube used is 50-100 kDa;

[0022] Preferably, the AD@PSSP needs to be stored at -80°C for future use.

[0023] The particle size of the AD@PSSP prepared by the present invention is 133-138nm, the dispersion index PDI is 0.21-0.25, the particle size is relatively uniform (PDI < 0.3), the nanoparticles within this range are stable, have lymph node enrichment effect, and are conducive to antigen presenting cell uptake, which can improve the targeting and bioavailability of antigens and adjuvants.

[0024] The PEI-SeSe-PEG aqueous solution is prepared by reacting PEI-SeSe-PEG powder with PBS in water, and the specific steps are: PEI-SeSe-PEG powder and PBS are evenly mixed at a ratio of 1-10 mg / ml to obtain a PEI-SeSe-PEG solution.

[0025] The preparation of the PEI-SeSe-PEG aqueous solution is carried out at room temperature, the stirring time is greater than or equal to 30 minutes, and the obtained PEI-SeSe-PEG aqueous solution is stored at 4°C;

[0026] Preferably, the incubation is: after uniformly mixing the aqueous solution containing PEI-SeSe-PEG and AD, the mixture is treated under ultrasound at room temperature; wherein the ultrasound time is 30-60 min and the room temperature is 20-25°C.

[0027] Furthermore, the ultrasonic power is 3-5w.

[0028] The present invention provides the use of the intravenous oncolytic virus capable of enhancing the therapeutic effect of RT in the preparation of a drug for treating malignant solid tumor diseases.

[0029] The present invention provides a pharmaceutical composition, comprising the intravenous oncolytic virus capable of enhancing the therapeutic effect of RT and pharmaceutically acceptable excipients.

[0030] The present invention provides application of the above-mentioned pharmaceutical composition in preparing medicine for treating malignant solid tumor diseases.

[0031] A combined treatment approach, including the above-mentioned AD@PSSP and RT;

[0032] Preferably, the single dose of RT is 2 Gy or more.

[0033] Furthermore, the above application is that AD@PSSP is used as a drug and RT to synergistically treat malignant solid tumor diseases.

[0034] Furthermore, the application is: intravenous injection of AD@PSSP and local RT are used simultaneously to treat malignant solid tumor diseases.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This patent successfully achieved the bonding of PSSP to the surface of AD through electrostatic interaction to prepare AD@PSSP nanoparticles. Compared with simple AD or AD-PEI, intravenous injection of AD@PSSP can significantly prolong its circulation time in the blood and improve safety. AD@PSSP can effectively release AD-PEI at the tumor site that receives radiotherapy and produces ROS. Compared with AD released alone, AD-PEI has enhanced infection ability and can capture endogenous tumor antigens produced by radiotherapy, thereby greatly enhancing the degree of tumor DNA damage and antigen-specific immune response after radiotherapy. Therefore, AD@PSSP not only effectively enhances the inhibitory effect of radiotherapy on systemic tumor growth, but also activates persistent immune memory, which helps to inhibit the recurrence of postoperative tumors.

[0037] This patent provides new ideas for the comprehensive treatment of cancer and brings new hope for achieving long-term control and cure of cancer. By combining radiotherapy and immunotherapy strategies, AD@PSSP has shown great potential in cancer treatment and is expected to become an effective anti-tumor treatment. In the future, this patent provides important support for conducting clinical trials based on this patent and further clinical applications. By further optimizing and improving the preparation method and use conditions of AD@PSSP, we hope to promote it to clinical application, thereby providing cancer patients with more personalized and effective treatment options. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A is the electron microscopy and particle size characterization of the AD tumor vaccine in Example 1, with a scale of 50 nm;

[0039] Figure 1 B is the electron microscopy and particle size characterization of the AD@PSSP tumor vaccine in Example 1, with a scale of 50 nm;

[0040] Figure 2 is the blood circulation time of AD and AD@PSSP in mice in Example 2;

[0041] Figure 3 In Example 3, the mean fluorescence intensity of DCF in the tumor was accurately quantified at 0, 2, 6, 12 and 24 hours after TC-1 tumor cells were given a 2Gy radiation dose;

[0042] Figure 4 In Example 3, the mean fluorescence intensity of DCF in the tumor was accurately quantified 12 hours after TC-1 tumor cells were given radiation doses of 0, 1, and 2 Gy;

[0043] Figure 5 It is the fluorescence intensity of each group of nanoparticles at a wavelength (550nm-650nm) in Example 3. G1: AD(BHQ2)@PSSP-Cy3, G2: DCH(80)+AD(BHQ2)@PSSP-Cy3, G3: DCH(220)+AD(BHQ2)@PSSP-Cy3, G4: DCH(350)+AD(BHQ2)@PSSP-Cy3, G5: AD@PSSP-Cy3;

[0044] Figure 6 In Example 4, after intravenous injection of AD@PSSP-Cy5.5, TC-1 tumors were taken for fluorescence imaging at 2h, 6h, 12h, and 24h;

[0045] Figure 7As in Example 5, C57BL / 6 mice bearing TC-1 tumors were intravenously injected with normal saline (G1), AD(BHQ2)@PSSP-Cy3 (G2), and RT (2Gy) + AD(BHQ2)@PSSP-Cy3 (G3), and TC-1 tumors and major organs were collected 24 hours later for ex vivo imaging;

[0046] Figure 8 The images of TC-1 tumors resected and immunofluorescence stained 24 hours after intravenous administration of different treatments in Example 6. Scale bar: 1 cm;

[0047] Fig. 9 is the survival fraction of HCT-116 cells under different drugs and irradiation in Example 7. Based on the results of the clone formation experiment, the D0 of different drugs was calculated using the multi-target impact model;

[0048] Fig.10 In Example 7, proteomic analysis of HCT-116 cells (irradiated with 6 Gy, 3 times in total) treated with different drugs for 24 hours revealed differentially expressed proteins. These proteins may lead to increased RT sensitivity, and we analyzed the relative abundance differences of these proteins between the PBS group and the AD-PEI group;

[0049] Fig.11 This is an ex vivo fluorescent image of the draining lymph node in Example 8 (n=4);

[0050] Fig.12 A is the ratio of Cy5.5 positive cells in the draining lymph nodes analyzed by flow cytometry in Example 8 (n=3);

[0051] Fig.12 B is the proportion of antigen presenting positive cells in DC cells in draining lymph nodes analyzed by flow cytometry in Example 8 (n=3);

[0052] Fig.13 The tumor photos after treatment in Example 9 (n=5);

[0053] Fig.14 A is the AD content in the tumor after treatment in Example 9 (n=5);

[0054] Fig.14 B is the analysis of tumor CTL cells using flow cytometry after treatment in Example 9 (n=5);

[0055] Fig.15 This is the hematoxylin-eosin staining of the lung, liver, heart, kidney, and spleen after treatment in Example 9, scale bar, 50 μm (n=5);

[0056] Fig.16 A is the tumor growth weight of each group of mice after in vivo treatment in the nude mouse model in Example 10 (n=5);

[0057] Fig.16 B is a photo of the tumors of mice in each group after in vivo treatment in the nude mouse model in Example 10 (n=5);

[0058] Fig.17 In Example 11, in vivo bioluminescence imaging of the primary tumor of the postoperative animal model was performed before, after, and 20, 40, and 60 days after surgery (n=10);

[0059] Fig.18 A is the distant tumor growth curve of each group of mice after postoperative animal model treatment in Example 11 (n=5);

[0060] Fig.18 B is a photo of distant tumors of mice in each group after the combined surgery animal model treatment in Example 11 (n=5);

[0061] Fig.19 A is the T cells in blood cells analyzed by flow cytometry after the animal model was treated postoperatively in Example 11. em The proportion of cells in CTL cells (n=5);

[0062] Fig.19 B is the analysis of T cells in spleen cells by flow cytometry after the animal model was treated postoperatively in Example 11. em The proportion of cells in CTL cells (n=5); DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0064] In the following examples, the genetically engineered oncolytic virus AD expressing the telomerase (TERT) promoter and AD5 enhancer 11 It was a gift from Beijing Bio-Targeted Therapeutics Technology Co., Ltd. and Zhengzhou University.

[0065] In the following examples, PSSP was used, which was purchased from Xi'an Ruixi Biotechnology Co., Ltd. (China).

[0066] Example 1

[0067] In this embodiment, the capture AD@PSSP is constructed by the following method:

[0068] (1) 400 μL of PSSP solution (1 mg / mL) and 100 μL of AD (1×109 pfu / mL) and incubated under ultrasound at room temperature for 0.5 h. Centrifuge at 10,000 rpm at 4°C for 5-10 min, and use an ultrafiltration core to remove excess PSSP;

[0069] (2) using a 100 kDa ultrafiltration tube to ultrafilter the solution obtained in (1) to remove unreacted PSSP, dissolving the residue in the filter membrane with PBS to obtain the AD@PSSP solution, which was then stored at -80° for future use;

[0070] (3) The morphology and particle size of the obtained AD and AD@PSSP were characterized by transmission electron microscopy (TECNAI G220S-TWIN, 200 kV, FEI, USA) and laser particle size analyzer (ZetasizerNano ZS90, Malvern, UK). Figure 1 As shown, Figure 1 A is the transmission electron microscopy image of the naked AD tumor vaccine, with a particle size of approximately 100.4 nm and a dispersion index (PDI) of 0.0161; Figure 1 B is the prepared AD@PSSP, which is spherical and has a smaller edge than AD 11 Blurred, the particle size is relatively uniform, the particle size is about 134.8nm, and the dispersion index (PDI) is 0.2421; compared with the naked AD 11 In comparison, the particle size increased by about 35 nm. The change in electron microscopy morphology and the increase in particle size indicate that we have successfully prepared AD@PSSP.

[0071] Example 2

[0072] The purpose of this example is to verify that AD@PSSP can significantly prolong the blood circulation time of the virus. The method is:

[0073] (1) Mix 10ul NHS-Cy5.5 solution (0.052mg / ml) and 10ul PSSP-Cy5.5 solution (1mg / ml) with 100μL AD (1×10 9 pfu / ml) and incubated at room temperature under ultrasound for 5 h. The free Cy5.5-NHS was then dialyzed in phosphate buffer (molecular weight cutoff: 30,000) for 24 hours, and the residue in the dialysis bag was dissolved by PBS to obtain the AD-Cy5.5 solution and AD@PSSP-Cy5.5 solution, which were then stored at -80° for later use.

[0074] (2) 100ul of saline, AD-Cy5.5 (1×10 8 pfu / mL) and AD@PSSP-Cy5.5 (1×10 8pfu / mL) was intravenously injected into C57BL / 6 mice TC-1. 10ul of blood was collected from the tail vein after 2h, 6h, 12h, 24h, 48h, and 72h.

[0075] (3) The blood of each group at each time point was examined by in vitro fluorescence examination using the Maestro system (Caliper, USA, IVIS Spectrum). Figure 2 As shown, AD@PSSP can significantly prolong the blood circulation time of the virus compared with AD.

[0076] The cells of this example are non-small cell lung cancer (TC-1) cancer cell lines modified to express human CD46 receptor.

[0077] Example 3

[0078] The purpose of this example is to verify in vitro at what radiotherapy dose the SeSe bond in AD@PSSP can be broken.

[0079] (1) After giving 10 10 After 0 hours, 2 hours, 6 hours, 12 hours, and 24 hours of 2Gy radiation dose to TC-1 tumor cells, the cell suspension was extracted, and the ROS of each group of cells was quantified according to the instructions of the reactive oxygen species (ROS) detection kit (E004-1-1) (the average fluorescence intensity of DCF can be used as an indicator of the intracellular ROS level). Figure 3 As shown, the ROS level in tumor tissues after irradiation with different doses reached a peak at 12 h after RT.

[0080] (2) After giving 10 10 12 hours after TC-1 tumor cells were irradiated with 0, 1, and 2 Gy doses, the cell suspension was extracted and the ROS in each group of cells was quantified according to the instructions of the reactive oxygen species (ROS) detection kit (E004-1-1). Figure 4 As shown, the average fluorescence intensity of DCF under 0 Gy, 1 Gy and 2 Gy radiation treatments were 81.8, 219.3 and 345.8, respectively.

[0081] (3) NHS-BHQ2 and PEI-SeSe-PEG-Cy3 were mixed in a PBS solution at a molar mass ratio of 1:1. 6 / Vp ratio and the above mixture was mixed with the AD treatment agent for 30 minutes. Subsequently, the mixture was dialyzed in a phosphate buffer at 4°C, using a dialysis bag with a molecular weight cutoff of 10,000 to remove NHS-BHQ2 and PEI-SeSe-PEG-Cy3 that did not participate in the reaction, for 24 hours. The final product was AD(NHS-BHQ2)-@PSSP-Cy3. Relatively speaking, the corresponding formula AD@PSSP-Cy3 was prepared by mixing NHS-PEG1Kda and PEI-SeSe-PEG-Cy3 in a 1:1 ratio with an equimolar ratio.

[0082] (4) We prepared H2O2 solutions of different concentrations to simulate the ROS levels observed in tumors after receiving 0Gy, 1Gy, and 2Gy RT, where the average fluorescence intensity of 2',7'-dichlorofluorescein corresponds to 80, 220, and 350, respectively. After AD(BHQ2)@PSSP-Cy3, AD@PSSP-Cy3, and AD(BHQ2)@PSSP-Cy3 were immersed in the above H2O2 solutions for 10 minutes, they were placed in a fluorescence spectrophotometer (Hitachi, Japan, Model F-4600) to measure their respective spectral characteristics. Figure 5 As shown, when the average fluorescence intensity of DCF reached 350, the fluorescence was completely restored, suggesting that the minimum radiation dose required for the shedding of PEG shell in vivo should be 2 Gy, which is also the low dose commonly used in clinical radiotherapy.

[0083] The radiotherapy in this embodiment was performed by an X-ray biological irradiator (Rod Source technologies Asia Limited, USA, RS2000).

[0084] The cells of this example are non-small cell lung cancer (TC-1) cancer cell lines modified to express human CD46 receptor.

[0085] Example 4

[0086] The purpose of this example is to verify the duration of combined treatment of RT and AD@PSSP.

[0087] 100ulAD@PSSP-Cy5.5 (1×10 8 pfu / ml) were intravenously injected into C57BL / 6 mice TC-1. Tumors were removed from each group after 2h, 6h, 12h, and 24h, and the tumors at each time point were examined by in vitro fluorescence using the Maestro system (Caliper, USA, IVIS Spectrum). Figure 6As shown in Figure 3, after intravenous injection of AD@PSSP-Cy3, the accumulation of AD in the tumor reached a maximum at 12 hours after injection, which coincided with the peak time of ROS level at a radiation dose of 2 Gy. Therefore, the optimal combined treatment time is to perform RT and AD@PSSP intravenous injection treatment simultaneously.

[0088] Example 5

[0089] The purpose of this example is to verify in vivo that the SeSe bond in AD@PSSP can be broken under tumor radiotherapy.

[0090] 100ulAD(BHQ2)@PSSP-Cy3(1×10 8 pfu / ml) probes were delivered intravenously to C57BL / 6 mice bearing TC-1 tumors. One group of mice received 2Gy of tumor RT, while the other group did not receive any radiation. After 24 hours, tumors, liver, spleen, kidney, lung, and heart tissues were dissected and analyzed by ex vivo fluorescence imaging using the Maestro system (Caliper, USA, IVIS Spectrum model). Figure 7 As shown in Figure 2, fluorescence recovery was observed in tumors after RT of mice injected with AD-BHQ-2@PSSP-Cy3, indicating that the SeSe bonds in AD@PSSP can be broken under tumor radiotherapy. In addition, we did not observe significant fluorescence recovery in major organs such as liver, kidney, spleen, heart, and lung of AD-BHQ-2@PSSP-Cy3 mice after RT, indicating that the SeSe bonds in PSSP do not break in these important organs.

[0091] Example 6

[0092] The purpose of this example is to verify in vivo whether AD@PSSP can restore the infection function after SeSe breaks.

[0093] Next, we further aimed to confirm whether PEI was still attached to AD and whether PEI maintained its basic function after the SeSe bond was broken. Different treatments were given intravenously to TC-1 tumor-bearing C57BL / 6 mice. After 24 hours, the TC-1 tumors were removed and immunofluorescence staining was performed. The specific scheme is as follows: According to Example 1, AD@PEG, AD@SSP, and AD@PSSP were prepared for use. 100ul of saline, AD@PEG (1×10 8 pfu / ml),AD@SSP(1×10 8 pfu / ml),AD@PSSP(1×10 8pfu / ml) was intravenously injected into C57BL / 6 mice. At the same time as the intravenous injection, 2 Gy radiotherapy was given to subcutaneous TC-1 tumors. 24 h later, the TC-1 tumors were collected for rapid frozen sectioning using the Maestro system (Caliper, USA, IVIS Spectrum). The sections were stained with DAPI to show the cell nuclei and examined using a laser scanning confocal microscope (3DHISTECH, Hungary, Panorama MIDI, Panorama 250FLASH, Panorama DESK). AD expressed GFP, and TC-1 cells infected with AD appeared GFP positive. Figure 8 The RT+AD@PSSP group has the strongest GFP fluorescence, which means that the tumor cells are infected with the most viral genes. In short, AD@PSSP can effectively respond to the ROS generated by RT, achieve the aggregation and targeted release of viruses at the tumor site, and effectively restore the infection ability of AD due to the timely shedding of the PEG shell, and the presence of PEI further enhances the infectivity of AD.

[0094] Example 7

[0095] The purpose of this example is to verify the mechanism of DNA damage after AD-PEI enhanced RT.

[0096] RT mainly irradiates tumors through high-energy radiation, acts on cancer cell DNA, causes damage and leads to cell death. AD-PEI can not only "dissolve" and infect tumor cells, but also enhance the RT effect by damaging cancer cell DNA. We treated HCT-116 cells with different reagents (PBS, PEI, AD and AD-PEI) and conducted clone formation experiments at different radiation doses (0Gy, 3Gy, 6Gy and 9Gy). The specific scheme is as follows: HCT-116 cells were inoculated in 6-well plates, with approximately 300 cells per well, and the cells were treated with PBS, AD, PEI and AD-PEI (dose of 1pfu / cell). Each treatment group was then subjected to different doses of X-ray radiation, with radiation doses of 0, 3, 6, and 9Gy, respectively. After 24 hours of radiation treatment, fresh culture medium was replaced, and cell growth was observed regularly. When the cells in the PBS-treated and non-irradiated group (0Gy) formed obvious cell colonies, the culture was terminated. Then discard the culture medium, wash the cells three times with PBS, and then leave them to air dry at room temperature. Add 1 mL of methanol to each well to fix the cells for 15 minutes, then pour off the methanol and let the plate air dry again. Use crystal violet solution to stain the cells for 15 minutes, rinse gently with tap water after staining, remove excess staining solution, and then leave the plate to air dry naturally. Analysis is performed by counting the number of colonies formed in each well of a 6-well plate (each colony consists of more than 50 cells). The multi-target hit model was used to fit the survival curve of the acquired data to calculate the biological parameters related to radiation. The calculation formula for the survival fraction (SF) is SF=1-(1-e(-D / D0))N, where D0 represents the mean lethal dose, D represents the actual radiation dose, and N represents the extrapolated number. Through this model, we further explored the effect of radiation dose on the survival of HCT-116 cells. Fig. 9 As shown, HCT-116 cells treated with AD-PEI had the highest radiosensitivity, with D0 reduced from 3.675 Gy in the PBS group and 2.732 Gy in the AD group to only 1.580 Gy.

[0097] We explored the mechanism by which AD-PEI enhances the radiosensitivity of tumor cells. First, HCT-116 cells were irradiated multiple times (6 Gy, 3 times), and then treated with PBS and AD-PEI, and proteomic analysis was performed. The specific scheme is as follows: The experimental raw data were obtained through protein extraction, quantification, quality control detection, restriction endonuclease digestion and desalting, component separation and mass spectrometry analysis. Subsequently, these data files were processed and analyzed in Proteome Discovserer version 2.2 software (Sequent HT, Thermo Fisher Scientific) and matched to the Uniprot protein database of Mus musculus (version date is January 27, 2022, containing 86515 sequences). The relative abundance of AD11-Mal-enriched proteins was calculated by dividing the standardized abundance value of each protein in the sample by the standardized abundance value in TC-1 tumor cells. To ensure the accuracy and reliability of the analysis results and reduce the probability of false positives, the Proteome Discovserer software strictly screened the searched results. Only peptide spectrum matches with a confidence level of more than 99% were considered reliable peptide spectrum matches, and only proteins containing at least one unique peptide were considered reliable proteins. During the data processing, we only retained these peptides and proteins that were verified to be reliable, and performed a false discovery rate check on them, excluding all peptides and proteins with a false discovery rate higher than 1%, thereby ensuring the high quality and scientific nature of the experimental analysis results. Fig.10 As shown, compared with the PBS group, the AD-PEI-treated group had 195 proteins with increased expression and 76 proteins with decreased expression, of which 20 proteins had been reported to enhance the effect of RT in previous studies.

[0098] The cells of this example are human colon cancer (HCT-116) cancer cell lines.

[0099] Example 8

[0100] The purpose of this example is to verify whether AD@PSSP can drain tumor antigens to lymph nodes after radiotherapy and whether it promotes antigen presentation.

[0101] After RT, it is extremely important for tumor antigens to be drained to lymph nodes to initiate anti-tumor immune responses. To determine whether AD-PEI can drain tumor antigens to lymph nodes in vivo, we inoculated TC-1 cells into the dorsal right thigh of C57BL / 6 mice. 100ul Cy5.5-labeled OVAs (1mg / ml) were injected into the surrounding tissues of TC-1 tumors in C57BL / 6 mice. Afterwards, mice were intravenously injected with 100ul saline, AD@SSP (10 7pfu / ml) or AD@PSSP (10 7 pfu / ml). At the same time, local tumor irradiation of 2Gy was performed. After 24 hours, the draining lymph nodes were collected from the right side of the mice and analyzed by in vitro fluorescence imaging using the Maestro system (Caliper, USA, IVIS Spectrum model). A flow cytometer (Accuri C6, BD, USA) was used to evaluate the Cy5.5-positive cells in DCs and their antigen-presenting ability. The specific steps are as follows: First, a 70μm cell strainer was used to crush the lymph nodes into a single cell suspension. After crushing, centrifuge at 1500rpm for 5 minutes at room temperature to precipitate the cells, and then the supernatant was discarded. Then, the cell pellet was resuspended in RPMI 1640 culture medium containing 2% fetal bovine serum, and the proportion of Cy5.5-positive cells in the lymph nodes was analyzed by flow cytometry (performed according to the operating instructions in the machine) to evaluate antigen uptake. Antigen presentation was measured using PE / Cy7-labeled anti-mouse H-2Kb (114616, clone name: 28-8-6) antibody that binds to SIINFEKL.

[0102] like Fig.11 As shown in the figure, the strongest Cy5.5 fluorescence signal was detected in the lymph nodes of the RT+AD@PSSP group. Fig.12 Flow cytometry analysis of lymph node cells as shown in A and 12B showed that the RT+AD@PSSP group contained the most Cy5.5-positive cells and DC cells for antibody presentation. This further demonstrates that AD-PEI can effectively drain tumor antigens released by RT to lymph nodes and present more antigens to T cells.

[0103] Example 9

[0104] The purpose of this example is to verify the anti-tumor efficacy, anti-tumor mechanism and safety of RT combined with AD@PSSP.

[0105] Considering the therapeutic effects of AD@PSSP and RT in vitro, we further evaluated its anti-tumor ability in the TC-1-loaded C57BL / 6 mouse model with pre-existing AD immunity. The specific experimental scheme is as follows: In the construction of the tumor model, first, on day 0, 3.5×10 6 TC-1 tumor cells. On days 7, 9, 11, and 13, the primary tumors were injected with various drugs (including AD, AD@SSP, and AD@PSSP, with a dose of 5×10 7pfu). The RT cycle was 8 days (from day 7 to day 14), once a day, a total of 8 times, each dose was 2Gy. On day 21, the primary tumor, distant tumor, spleen and serum of the mice were analyzed. During the administration period, the tumor volume and body weight were measured every two days. The calculation formula of V is: V = 1 / 2ab 2 , where "a" is the long axis of the tumor and "b" is the short axis of the tumor.

[0106] like Fig.13 As shown, in the cohort without RT, enhanced therapeutic effects were observed in the group with the presence of PEG component compared to AD, highlighting the role of PEG in avoiding neutralizing antibodies and prolonging drug circulation time. The best therapeutic effect was achieved in the AD@PSSP treatment group, which may be attributed to the intrinsic ROS in the tumor promoting the responsive release of AD@PSSP. In the treatment group that received RT, mice combined with AD@PSSP showed the most significant synergistic effect.

[0107] To further understand the anti-tumor mechanism, we analyzed the tumor samples after treatment. Fig.14 As shown in A and B, the accumulation of OVs in the tumors of the RT+AD@PSSP treatment group was the most significant. In the RT+AD@PSSP group, cytotoxic T lymphocytes (CD3 + CD8 + The above results indicate that the therapeutic effect of RT+AD@PSSP group is mainly mediated by viral infection and antigen adaptive immune killing.

[0108] After the treatment, the lungs, livers, hearts, kidneys, and spleens of the mice were removed for hematoxylin-eosin staining. The specific experimental steps were as follows: Paraffin sections were made for each organ. Then the sections were placed in xylene I for 10 min-xylene II for 10 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-95% alcohol for 5 min-90% alcohol for 5 min-80% alcohol for 5 min-70% alcohol for 5 min-distilled water. Then the sections were stained with Harris hematoxylin for 3-8 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water. The sections were stained with eosin staining solution for 1-3 min. Finally, the sections were placed in 95% alcohol I for 5 min-95% alcohol II for 5 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min, xylene I for 5 min-xylene II for 5 min, dehydrated and transparent, and the sections were taken out of xylene to dry slightly and sealed with neutral gum. Microscope examination, image acquisition and analysis. Fig.15 As shown, no pathological damage was found in major organs during the entire treatment process.

[0109] Example 10

[0110] The purpose of this example is to verify the anti-tumor efficacy of RT combined with AD@PSSP in an immunodeficient nude mouse model.

[0111] We further verified the synergistic therapeutic effect of AD@PSSP and RT in an immunodeficient mouse model (BALB / c nude mice) without immunity. The specific scheme is as follows: In BALB / c nude mice, 1×10 8 HCT-116 cells were treated with the corresponding drugs on days 12 and 14. RT was performed on days 12 to 15 for 4 consecutive days, once a day, with a dose of 2 Gy each time. Tumors were collected on day 21. Fig.16 As shown in A and B, compared with other groups, the RT+AD@PSSP group showed more obvious effects in inhibiting tumor growth.

[0112] Embodiment 11

[0113] The purpose of this example is to verify whether RT combined with AD@PSSP treatment can induce long-term immune memory.

[0114] Tumor recurrence after surgery is a major challenge in clinical cancer treatment. Therefore, RT is usually used after surgery to eliminate any residual potential malignant cells and reduce the risk of local recurrence or distant metastasis. We further evaluated the potential of AD@PSSP as an adjuvant therapy for postoperative RT, especially in inducing persistent immune memory. The evaluation method is as follows: 3.5×10 6 TC-1-Luc cells were inoculated subcutaneously on the right back of C57BL / 6 mice, and a primary tumor model was established on the 20th day. Three days later, the tumor was surgically removed, leaving 10% of the residual tumor. After that, different drugs were given 4 times by intravenous drip on days 0, 2, 4, and 6. RT was performed daily from day 0 to day 7, divided into 8 times, 2 Gy each time. In vivo imaging of mice was performed before and after surgery, and on days 20, 40, and 60 after surgery. The in vivo imaging analysis method is as follows: in PBS (without Mg 2+ and Ca 2+ ) was used to prepare a 15 mg / mL luciferin working solution, and the bacteria were removed by filtering through a 0.2 μm filter membrane. The injection volume was calculated as 10 μL / g body weight. 10 to 15 minutes after the intraperitoneal injection, the mice were placed on an imaging machine for analysis. The growth of the primary tumor volume was measured every 1 day until the 60th day. Fig.17 As shown, 80% of C57BL / 6 mice treated with RT+AD@PSSP survived, while the survival rate of C57BL / 6 mice treated with postoperative RT alone was only 30%. On day 60, 3.5×10 6TC-1-Luc cells were used to establish distant tumors, and healthy mice of the same age were used as controls. Fig.18 As shown in A and B, C57BL / 6 mice treated with RT+AD@PSSP showed significantly reduced tumor growth compared with the control group.

[0115] On day 80, we evaluated immune memory cells in splenocytes and blood by flow cytometry. Fig.19 As shown, compared with the control group, C57BL / 6 mice treated with RT+AD@PSSP had a higher proportion of immune memory T cells in splenocytes and blood. em (CD3 + CD8 + CD44 + CD62L - ).

[0116] The cells of this example are non-small cell lung cancer cell lines (TC-1-Luc) that have been modified to express human CD46 receptor and Luc gene.

Claims

1. An intravenous oncolytic virus capable of enhancing the efficacy of radiotherapy, characterized in that: The intravenous injection type oncolytic virus that can enhance the efficacy of radiotherapy expresses a genetically engineered oncolytic adenovirus AD5 that is a telomerase promoter and an AD5 enhancer. 11 .

2. The oncolytic virus according to claim 1, characterized in that The oncolytic virus modification group is polyethyleneimine-diselenoic acid-polyethylene glycol.

3. The method for preparing an intravenous oncolytic virus capable of enhancing the therapeutic effect of radiotherapy according to any one of claims 1 to 2, characterized in that: OVs were incubated with an excess of PEI-SeSe-PEG at room temperature, and then the unreactive active groups were removed through a filter membrane and an ultrafiltration tube. After PBS was added again and resuspended in the ultrafiltration tube, the intravenous oncolytic virus capable of enhancing the efficacy of radiotherapy was obtained.

4. The method for preparing an intravenous oncolytic virus capable of enhancing the therapeutic effect of radiotherapy according to claim 3, characterized in that: The PEI-SeSe-PEG solution was mixed with AD and then incubated at room temperature to promote full binding between the two. Then, the unreactive active groups were removed by filtration membrane and ultrafiltration tube, and PEG was stained with barium iodide to determine the maximum amount of the reaction preparation; among them, AD was 11 :PEI-SeSe-PEG(1.6×10 6 / vp).

5. The method for preparing an intravenous oncolytic virus capable of enhancing the therapeutic effect of radiotherapy according to claim 3, characterized in that: The molecular weight of PEG in the PEI-SeSe-PEG is 20Kda; in the ultracentrifuge tube, the low-speed centrifugation speed used is 5000-10000rpm, and the centrifugation is 5-10min; the pore size of the filter membrane used is 0.45μm and 0.22μm; and / or the molecular weight cutoff of the ultrafiltration tube used is 50-100kDa.

6. The method for preparing an intravenous oncolytic virus capable of enhancing the therapeutic effect of radiotherapy according to claim 3, characterized in that: The incubation is as follows: after uniformly mixing an aqueous solution containing PEI-SeSe-PEG with AD, the solution is treated under ultrasound at room temperature; wherein the ultrasound time is 30-60 min, and the room temperature is 20-25°C; the aqueous solution of PEI-SeSe-PEG is prepared by reacting PEI-SeSe-PEG powder with PBS in water, and the specific steps are as follows: uniformly mixing PEI-SeSe-PEG powder with PBS at a ratio of 1-10 mg / ml, and reacting in water.

7. The intravenous oncolytic virus capable of enhancing the therapeutic effect of radiotherapy according to any one of claims 1-2, characterized in that: The particle size of the intravenous oncolytic virus capable of enhancing the therapeutic effect of radiotherapy is 133-138 nm, and the dispersion index PDI is 0.21-0.

25.

8. Use of the intravenous oncolytic virus capable of enhancing the therapeutic effect of radiotherapy according to any one of claims 1-2 in the preparation of a drug for treating malignant solid tumor diseases.

9. A pharmaceutical composition comprising the intravenous oncolytic virus capable of enhancing the therapeutic effect of radiotherapy as claimed in any one of claims 1-2 and pharmaceutically acceptable excipients.

10. Use of the pharmaceutical composition according to claim 9 in the preparation of drugs for treating malignant solid tumors.

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