Intravenous oncolytic virus capable of enhancing radiotherapy efficacy, and preparation method therefor and use thereof

Abstract

The present invention relates to the field of nanomedicine. Disclosed are an intravenous oncolytic virus capable of enhancing the radiotherapy efficacy, and a preparation method therefor and a use thereof. In the present invention, PEI-Se-Se-PEG is used to modify the surface of a negatively charged oncolytic virus, thereby developing a "stealth" oncolytic virus suitable for intravenous injection, referred to as AD@PSSP. Compared with the prior art, intravenous injection of AD@PSSP can significantly prolong the circulation time of the oncolytic virus in blood and improve the safety; moreover, the inhibitory effect of radiotherapy on the growth of tumors of whole body is effectively improved, and long-lasting immunological memory can also be activated, thereby facilitating the inhibition of tumor recurrence.

Description

A venous injection type oncolytic virus capable of enhancing the effect of radiotherapy, and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of nanomedicine, and particularly relates to a venous injection type oncolytic virus capable of enhancing the effect of radiotherapy, and a preparation method and application thereof. BACKGROUND

[0002] Oncolytic viruses (OVs) represent a new immunotherapy approach. In recent years, significant progress has been made in clinical trials of melanoma and glioma. However, for patients with diffuse or recurrent tumors, it is often necessary to combine radiotherapy (RT) to improve the therapeutic effect. However, when these related clinical trials are carefully examined, there are still some problems to be solved. First, the antigen utilization rate of OVs is low, and a large amount of antigen is 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 it seems to be a better approach to treat metastatic tumors and tumors from deep organs by intravenous infusion of OVs, in clinical practice, the effect of intravenous administration is far from expected. Once OVs enter the blood circulation, the heterogeneity of the surface antigen proteins can activate the body's own immune system, causing antibodies and complements in the peripheral blood to bind to the virus to form immune complexes and promote the endocytosis of the virus 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 administration routes to expand the application range of OVs therapy. At the same time, efforts are needed to find ways to amplify the ability of OVs to remove tumor cells in combination with RT, such as enhancing the infection ability of OVs to strengthen the DNA damage effect and the ability of specific immune removal of tumors, which are challenges to be overcome.

[0003] Chemical modification and bio-membrane encapsulation have been proven 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 widely used for its excellent nucleic acid condensing and stabilizing 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 proven to be more effective than other commonly used cationic polymers such as chitosan and polyamide. PEI-nucleic acid complexes can be reproducibly prepared by simple mixing. In addition, PEI polyplexes 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, branched and linear PEI. At low molecular weight, PEI is often cross-linked with biodegradable linkers to maintain excellent nucleic acid condensing properties while improving biocompatibility. Branched PEI is synthesized by ring-opening polymerization of aziridine, while linear PEI is obtained by modification of other polymers such as poly(2-oxazoline). Both synthesis methods have been described in detail in the prior art and provide an efficient and cost-effective approach for large-scale production. PEI vectors have multi-functional properties due to the widespread presence of amine groups, adapting to the needs of various gene delivery.

[0004] Adenovirus (AD virus) 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 coated with bile acid conjugated PEI (AD / DA3) (Lee, Cho-Hee, et al. "Enhanced therapeutic efficacy of an adenovirus-PEI-bile-acid complex in tumors with low coxsackie and adenovirus receptor expression." Biomaterials 35.21 (2014): 5505-5516.). AD / DA3 significantly improves the infection efficiency in CAR medium or CAR negative cancer cells. Compared with naked AD, AD / DA3 significantly enhances apoptosis, reduces angiogenesis, reduces cell proliferation, and enhances active virus replication 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-limited tumors.

[0005] Since PEI is positively charged and tumor antigens are usually negatively charged, PEI has the potential to capture negatively charged antigens. AD-PEI (AD modified with PEI) that fails to enter tumor cells in time also has the potential to drain captured antigens to lymph nodes, triggering specific immune killing. In addition, it is important that PEI can also act as a carrier for antigen delivery, enhancing innate and adaptive immune responses, showing the characteristics of an immunological adjuvant. Endosomes containing proteases are responsible for intracellular sorting, and endosomal escape of vaccine complexes is essential for efficient antigen release. Degradation by lysosomes can hinder efficient release. The pH value in endosomes decreases from the physiological pH value of 7.4 in early endosomes to the pH value of 5.0 in lysosomes due to the action of membrane-bound ATPase pumps. PEI exhibits high buffering capacity at almost all pH values, so it is a compound with high endosomal escape activity. After endosomal acidification, cationic PEI can cause endosome rupture through the "proton sponge" effect; protonation causes passive influx of chloride ions, followed by influx of water, ultimately leading to osmotic swelling and endosome rupture, allowing PEI-based vaccines to escape to the interior of the vesicle. After the antigen in the PEI-based vaccine escapes from the endosome, it triggers a specific immune response of T cells through a cross-presentation mechanism. In addition, PEI-based vaccines have immunoreactivity that promotes systemic immune responses. Through in vivo immunization studies, it was found that PEI can activate CD8 and CD4 T cells in mixed Th1 / Th2 immune responses. However, the specific mechanism of action of PEI immunization is not clear. The immunological activity of PEI can be attributed to the activation of various molecular pathways, such as the TLR pathway, the NLRP3 inflammasome pathway containing the Pyrin domain, and the danger signal 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 immune killing after RT by capturing and presenting antigens.

[0006] However, the large number of positive charges carried by PEI is not conducive to intravenous injection. In addition, the use of polymer methods such as PEG has significantly prolonged the blood circulation half-life of drugs and improved their biocompatibility. Moreover, encapsulating drugs in biological membranes can optimize their distribution in the body and achieve homologous targeting by taking advantage of the natural homing properties of living organisms. Therefore, PEGylation or biological membrane modification of OVs-PEI can facilitate the intravenous injection application of the complex. However, as a biologically active entity, OVs can suffer significant loss of infection performance when subjected to chemical modification or wrapped in biological membranes. Therefore, the biggest challenge in intravenous infusion of OVs-PEI is to develop a strategy that can remove the protective layer after reaching the tumor microenvironment, thereby restoring the original infection performance of the virus.

[0007] Controlled release of nanomedicines, with its responsiveness, not only ensures the precise delivery of the targeted drug, but also precisely deconstructs the protective nanoshell through the rearrangement of molecular structure upon receiving the activation signal. Such responsive systems are designed to take advantage of the special physicochemical properties inherent in the tumor microenvironment, such as enhanced permeability, acidic pH, or overexpressed enzymes. In the exogenous aspect, the introduction of external excitation factors such as magnetic field, focused ultrasound or RT further expands the strategy library of nanomedicines in precise navigation and drug release. In particular, the synthesis of reactive oxygen species (ROS)-responsive polymer carriers and their medical applications have attracted extensive research interest. Numerous ROS-responsive polymers for drug delivery systems have been developed and shown advantages over traditional drug delivery methods in therapeutic effect. Currently, various types of ROS-responsive polymer carriers have been explored, including thioether-containing polymers, selenium (Se) / tellurium-containing polymers, arylboronic acid / ester-containing polymers, aryl oxalate and polyproline, etc. These ROS-responsive polymers usually cause chemical bond breakage and / or change from hydrophobic to hydrophilic phase, which helps to release the drug in the carrier. Se-containing polymers are of particular interest, as Se is an essential element for the human body and plays a key role in protecting cells from oxidative damage. Deficiency of Se in the human body can lead to Keshan disease and depression. Due to its high atomic number and low electronegativity, 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, C-S bond 272 kJ / mol, S-S bond 240 kJ / mol). Se-containing polymers are more sensitive to ROS, which can be oxidized to Se and selenone, changing from hydrophobic to hydrophilic through a mechanism similar to thioether. After oxidation of the diselenide bond to seleninic acid, it can be reduced to selenol and finally broken, similar to the reaction mechanism of disulfide bonds. Se-containing polymers are more sensitive to oxidants than sulfur-containing polymers 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 compounds and diselenide bonds are not stable in the presence of oxygen and the solubility of the polymers is poor. However, a groundbreaking study by Xu and Zhang et al. in 2010 showed that the solubility of Se-containing polymers could be improved by introducing diselenide groups into the diol structure. They synthesized amphiphilic diselenide polyurethanes by stepwise polymerization using toluene diisocyanate and diselenide-containing diols as raw materials. Since then, many Se-containing copolymers have been developed, leading to the synthesis of a large number of ROS-responsive polymer carriers.

[0008] SUMMARY

[0009] In view of the deficiencies of the prior art, the application provides a intravenous injection type OVs capable of enhancing the effect of RT, and a preparation method and application thereof, and develops a "stealth" OVs (named AD@PEI-SeSe-PEG, abbreviated as AD@PSSP) suitable for intravenous injection to cooperate with RT. The PEI-SeSe-PEG modifies the negatively charged OVs to resist neutralizing antibodies and prolong blood circulation. When the AD@PSSP circulates to the ROS generated after RT around the tumor, the Se-Se bond in the AD@PSSP breaks, the PEG falls off, and the infection ability of the OVs is restored. In addition, the de-PEGylated PEI can enhance the ability of the OVs to infect tumor cells through electrostatic interaction. In addition, the inventor observes that the OVs can weaken the activation of the CHEK1-CDK pathway, thereby enhancing the DNA damage after radiotherapy. Finally, AD-PEI as an in situ tumor vaccine carrier, captures tumor antigens after RT and delivers them to dendritic cells, which can produce an antigen-specific immune response, effectively ablate distant tumors, and be accompanied by long-lasting immune memory.

[0010] The OVs modification strategy described in the application is expected to lay a foundation for clinical trials involving OVs combined with RT and subsequent clinical deployment. Moreover, the OVs also have the characteristics of good biocompatibility, strong stability, high safety, high bioavailability and the like.

[0011] To achieve the object of the application, the application adopts the following technical solutions:

[0012] The application provides a intravenous injection type OVs-AD@PSSP capable of enhancing the effect of RT.

[0013] The OVs are preferably genetically engineered oncolytic adenovirus AD 11 .

[0014] The group is polyethylenimine-diselenide-polyethylene glycol (PEI-SeSe-PEG or PSSP)

[0015] The application provides a preparation method of AD@PSSP, which comprises the following steps:

[0016] The OVs (AD 11 ) are incubated with excess PEI-SeSe-PEG at room temperature, then the unreacted active groups are removed by an ultrafiltration tube, and after resuspension in the ultrafiltration tube, AD@PSSP is obtained.

[0017] Preferably, the preparation method of AD@PSSP is: mixing PEI-SeSe-PEG solution with AD, and then incubating at room temperature to promote sufficient combination between the two. Then, the unreacted active groups are removed by ultrafiltration tube.

[0018] The ratio of PEI-SeSe-PEG to AD is 1:1.6x10 11 Vp or above, which can ensure that PEI-SeSe-PEG is in excess. 6

[0019] Preferably, in PEI-SeSe-PEG, the molecular weight of PEG is 20 kDa or more, and our pre-experiment and related literature report that the resistance of PEGylated AD to neutralizing antibodies and macrophage phagocytosis increases with the increase of PEG molecular weight, and reaches the highest at 20 kDa.

[0020] Preferably, in the ultrafiltration tube, the low-speed centrifugal revolution is 5000-10000 rpm, and the centrifugation time is 5-10 min;

[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℃ for standby use.

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

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

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

[0026] Preferably, the incubation is: mixing the PEI-SeSe-PEG aqueous solution (i.e. PEI-SeSe-PEG solution) containing PEI-SeSe-PEG with AD 11 ​After mixing evenly, ultrasonic treatment is carried out at room temperature; wherein, the ultrasonic time is 30-60 min, and the room temperature is 20-25 DEG C.

[0027] Further, the ultrasonic power is 30-40 Hz, the ultrasonic time is 5 s of starting and 5 s of stopping.

[0028] The application provides application of the intravenous injection type oncolytic virus capable of enhancing the RT effect in preparation of a drug for treating a malignant solid tumor disease.

[0029] The application provides a pharmaceutical composition comprising the intravenous injection type oncolytic virus capable of enhancing the RT effect and a pharmaceutically acceptable excipient.

[0030] The application provides application of the above pharmaceutical composition in preparation of a drug for treating a malignant solid tumor disease.

[0031] Further, the application also provides a combined treatment method, comprising administering the intravenous injection type oncolytic virus capable of enhancing the RT effect to a subject before, simultaneously or after administering RT to the subject.

[0032] Preferably, in the combined treatment method, the single dose of the RT is above 2 Gy.

[0033] Preferably, in the combined treatment method, the administration dose of the intravenous injection type oncolytic virus capable of enhancing the RT effect is (1x10 7 pfu-5x10 7 pfu).

[0034] Preferably, the subject has a malignant solid tumor disease, including but not limited to colon cancer, lung cancer and glioma.

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

[0036] The present application successfully realizes the bonding of PSSP and AD surface through electrostatic action, and prepares AD@PSSP nanoparticles. Compared with pure AD or AD-PEI, AD@PSSP can significantly prolong the circulation time in the blood and improve the safety through intravenous injection. At the tumor site treated by radiotherapy and producing ROS, AD@PSSP can effectively release AD-PEI. Compared with the released AD alone, AD-PEI is enhanced in the infection ability and can capture the endogenous tumor antigens generated by radiotherapy, thereby greatly enhancing the degree of tumor DNA damage after radiotherapy and the antigen-specific immune response. Therefore, AD@PSSP not only effectively improves the inhibition effect of radiotherapy on the growth of whole body tumor, but also activates the long-lasting immune memory, which helps to inhibit the recurrence of postoperative tumor.

[0037] The present application provides a new idea for the comprehensive treatment of cancer and brings new hope for the long-term control and cure of cancer. By combining radiotherapy and immunotherapy strategies, AD@PSSP shows great potential in cancer treatment and is expected to become an effective anti-tumor treatment plan. In the future, the present application provides important support for carrying out clinical experiments and further clinical applications based on the present application. By further optimizing and improving the preparation method and use conditions of AD@PSSP, we hope to push it to clinical application, thereby providing more individualized and effective treatment options for cancer patients. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is the electron microscope and particle size characterization of AD naked in Example 1. 11 (A) and AD@PSSP (B), the scale is 50 nm.

[0039] Figure 2 is the blood circulation time of AD and AD@PSSP in mice in Example 2. 11

[0040] Figure 3 is the result of accurate quantification of the average fluorescence intensity of DCF in the tumor at 0, 2, 6, 12 and 24 hours after giving TC-1 tumor cells a radiation dose of 2Gy in Example 3.

[0041] Figure 4 is the result of accurate quantification of the average fluorescence intensity of DCF in the tumor at 12 hours after giving TC-1 tumor cells a radiation dose of 0, 1, 2Gy in Example 3.

[0042] Figure 5 is the fluorescence intensity curve of each group of nanoparticles at a wavelength (550nm-650nm) in Example 3. G1: AD(BHQ2)@PSSP-Cy3, G2: DCF(80)+G1, G3: DCF(220)+G1, G4: DCF(350)+G1, G5: AD@PSSP-Cy3. ​

[0043] Figure 6 is the result of fluorescence imaging of TC-1 tumor taken at 2h, 6h, 12h, 24h after intravenous injection of AD@PSSP-Cy5.5 in Example 4.

[0044] Figure 7 is the result of ex vivo imaging of TC-1 tumor and major organs collected 24h after intravenous injection of saline (G1), AD(BHQ2)@PSSP-Cy3 (G2) and RT (2Gy) + AD(BHQ2)@PSSP-Cy3 (G3) in Example 5.

[0045] Figure 8 is the image of TC-1 tumor excised and immunofluorescently stained 24h after intravenous administration of different treatment modalities in Example 6. Scale bar: 1cm.

[0046] Figure 9 is the survival fraction of HCT-116 cells under different drugs and irradiation in Example 7. According to the result of colony formation experiment, D0 of different drugs was calculated using the multi-target hit model.

[0047] Figure 10 is the proteomic analysis of HCT-116 cells (received 6Gy radiation, total of 3 times) treated with different drugs for 24h in Example 7 revealed differentially expressed proteins (exemplarily showed 20 of them). These proteins might lead to increased RT sensitivity, we analyzed the difference in relative abundance of these proteins between PBS group and AD-PEI group.

[0048] Figure 11 is the ex vivo fluorescence image of draining lymph nodes (n=4) in Example 8.

[0049] Figure 12 is the flow cytometry analysis of the proportion of Cy5.5 positive cells in draining lymph nodes (A) and the proportion of antigen presenting positive cells in antigen presenting cells in draining lymph nodes (B) (n=3) in Example 8.

[0050] Figure 13 is the photograph of tumor after treatment (n=5) in Example 9.

[0051] Figure 14 is the AD content in tumor after treatment (A) and the analysis of tumor CTL cells using flow cytometry after treatment (B) (n=5) in Example 9.

[0052] Figure 15 is the hematoxylin-eosin staining of lung, liver, heart, kidney, spleen after treatment (n=5) in Example 9, scale bar, 50μm.

[0053] Figure 16 shows the tumor growth weight (A) and tumor photographs (B) of each group of mice after in vivo treatment in the nude mouse model in Example 10 (n=5).

[0054] Figure 17 shows in vivo bioluminescence imaging of the primary tumor in the postoperative animal model before surgery, after surgery, and at 20, 40, and 60 days after surgery (n=10) in Example 11.

[0055] Figure 18 shows the distant tumor growth curves (A) and distant tumor photographs (B) of mice in each group after postoperative animal model treatment in Example 11 (n=5).

[0056] Figure 19 shows the analysis of T cells in blood cells (A) and spleen cells (B) by flow cytometry in the animal model after postoperative treatment in Example 11. em The proportion of cells in CTL cells (n=5). Detailed Implementation

[0057] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0058] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., 3rd edition, 2001, NY, Cold Spring Harbor).

[0059] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0060] In the following examples, genetically engineered oncolytic adenoviruses expressing the telomerase reverse transcriptase (TERT) promoter and AD5 enhancer were used. 11 It was a gift from Beijing Bio-Targeted Therapy Technology Co., Ltd. and Zhengzhou University.

[0061] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. For example, the PSSP used in the following examples was purchased from Xi'an Ruixi Biotechnology Co., Ltd. (China).

[0062] Example 1

[0063] In this example, AD@PSSP was constructed by the following method, which is:

[0064] (1) 400 μL of PSSP solution (1 mg / mL) was mixed with 100 μL of AD 11 (1 x 10 9 pfu / mL) and incubated for 0.5 h at room temperature under ultrasonic treatment (ultrasonic power was 35 Hz, ultrasonic start 5 s, stop 5 s);

[0065] (2) The solution obtained in (1) was subjected to ultrafiltration (centrifuged at 10000 rpm for 5-10 min at 4°C) using an ultrafiltration tube (filter membrane pore size was 100 kDa) to remove unreacted PSSP, and the residue in the filter membrane was dissolved with PBS to obtain AD@PSSP solution, which was stored at -80°C for standby;

[0066] (3) The morphology and particle size of AD 11 and the obtained AD@PSSP were characterized by transmission electron microscopy (FEI, Tecnai G2 20S-TWIN, 200 kV, USA) and laser particle size analyzer (Malvern, Zetasizer Nano ZS90, UK), as shown in Figure 1, wherein Figure 1A is the transmission electron micrograph of naked AD 11 tumor vaccine, the particle size was about 100.4 nm, and the dispersion index (PDI) was 0.0161; Figure 1B is the AD@PSSP prepared, which is spherical, the edge is more blurred than AD 11 , the particle size is more uniform, the particle size is about 134.8 nm, and the dispersion index (PDI) is 0.2421; compared with naked AD 11 , the particle size increases by about 35 nm, the change of electron microscope morphology and the increase of particle size indicate that AD@PSSP is successfully prepared in this example, which is a genetically engineered oncolytic adenovirus AD 11 obtained by using PSSP to modify the gene expressing telomerase reverse transcriptase (TERT) promoter and AD5 enhancer.

[0067] Example 2

[0068] The purpose of this example is to verify that AD@PSSP can significantly prolong the blood circulation time of the virus, and the method is:

[0069] (1) 10 μl NHS (N-Hydroxy succinimide)-Cy5.5 solution (Aladdin (China), 1469277-96-0) (0.052 mg / ml) and 10 μl PSSP-Cy5.5 solution (purchased from Xi'an Ruishi Biological Technology Co., Ltd.) (1 mg / ml) were mixed with 100 μL of AD 11 (1 x 10 9 pfu / ml) respectively, and incubated under ultrasonic at room temperature for 5 h. Then the free NHS-Cy5.5 and PSSP-Cy5.5 in both were removed by dialysis (molecular weight cutoff: 30000) in phosphate buffer. After 12-24 hours, the residues in the PBS dialysis bag were dissolved, and AD-Cy5.5 solution and AD@PSSP-Cy5.5 solution were obtained respectively, and stored at -80℃ for standby;

[0070] (2) 100 μl of saline, AD-Cy5.5 (1 x 10 8 pfu / mL) and AD@PSSP-Cy5.5 (1 x 10 8 pfu / mL) were injected intravenously into C57BL / 6 mice (purchased from Vantian Lihua Biological Technology Co., Ltd., 3 in each group) carrying TC-1 tumors respectively. 10 μl of blood was taken from the tail vein at 2 h, 6 h, 12 h, 24 h, 48 h, 72 h respectively.

[0071] (3) The blood of each group at each time point was examined in vitro for fluorescence using Maestro system (Caliper, USA, IVIS spectrum). The results are shown in Figure 2, which shows that AD@PSSP can significantly prolong the blood circulation time of the virus compared with AD.

[0072] Example 3

[0073] The purpose of this example is to verify in vitro how many doses of radiotherapy the SeSe bond in AD@PSSP can be broken.

[0074] (1) 10 10 TC-1 tumor cells (a modified non-small cell lung cancer (TC-1) cancer cell line expressing human CD46 receptor) were given a 2 Gy radiation dose at 0 h, 2 h, 6 h, 12 h, 24 h respectively, and cell suspensions were 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 (2', 7'-dichlorofluorescein) can be used as an indicator of intracellular ROS level). The results are shown in Figure 3, which shows that the ROS level in the tumor cells after 2 Gy irradiation reached a peak at 12 hours after RT.

[0075] (2) 10 Gy of radiation was given to the mice 10 TC-1 tumor cells were extracted 12 hours after 0, 1, 2 Gy radiation dose, 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 results are shown in Figure 4, which shows that the average fluorescence intensity of DCF under 0 Gy, 1 Gy and 2 Gy radiation treatment was 81.8, 219.3 and 345.8, respectively.

[0076] (3) NHS-BHQ2 and PEI-SeSe-PEG-Cy3 were mixed uniformly in PBS solution at a molar ratio of 1:1. Then, 1:1.6 x 10 6 The above mixture was mixed with AD 11 for 30 minutes at a ratio of Vp (the ratio of the number of solutes to the number of virus particles). Subsequently, the mixture was dialyzed in phosphate buffer at 4°C for 24 hours 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. The final product was named AD(BHQ2)@PSSP-Cy3. According to the same operation, the corresponding product was prepared by mixing NHS-PEG 1KDa and PEI-SeSe-PEG-Cy3 at a molar ratio of 1:1, and was named AD(PEG 1KDa)@PSSP-Cy3 (abbreviated as AD@PSSP-Cy3).

[0077] (4) We prepared H2O2 solutions with concentrations of 2%, 5%, and 10%, which were set to simulate the ROS levels observed in tumors after receiving 0 Gy, 1 Gy, and 2 Gy RT, with average fluorescence intensities of DCF corresponding to 80, 220, and 350, respectively. After soaking AD@PSSP-Cy3 and AD(BHQ2)@PSSP-Cy3 in the above H2O2 solutions for 10 minutes, we placed them in a fluorescence spectrophotometer (Japan Hitachi, model F-4600) to measure their spectral properties. The results are shown in Figure 5, which shows that when the average fluorescence intensity of DCF reaches 350, the fluorescence is completely restored, suggesting that the minimum radiation dose required for the shedding of the PEG shell in vivo should be 2 Gy, which is also a commonly used low dose in clinical radiotherapy.

[0078] The radiotherapy of this example was completed by an X-ray biological irradiator (Rod Source technologies Asia Limited, USA, RS2000).

[0079] Example 4

[0080] The purpose of this example is to verify the time of RT combined with AD@PSSP treatment.

[0081] 100 μl of AD@PSSP-Cy5.5 (prepared in Example 2) (1×10 8 (pfu / ml) was intravenously injected into C57BL / 6 mice carrying TC-1 tumors (n=3 per group). Tumors were removed from each group at 2h, 6h, 12h, and 24h, and in vitro fluorescence examination was performed using a Maestro system (Caliper, USA, IVIS spectroscopy). The results are shown in Figure 6. It can be seen that after intravenous injection of AD@PSSP-Cy3, the accumulation of AD in the tumor reached its maximum at 12 hours post-injection, consistent with the peak time of ROS levels at a 2Gy radiation dose. Therefore, the optimal time for combined treatment is simultaneous RT and intravenous injection of AD@PSSP.

[0082] Example 5

[0083] The purpose of this embodiment is to verify in vivo that the SeSe bond in AD@PSSP can be broken under tumor radiotherapy.

[0084] 100 μl of AD(BHQ2)@PSSP-Cy3 (prepared in Example 3) (1×10 8 (pfu / ml) was administered intravenously to C57BL / 6 mice carrying TC-1 tumors. One group of mice received 2 Gy tumor radiotherapy (RT), while the other group (n=3 per group) received no radiotherapy. Twenty-four hours later, the tumors, liver, spleen, kidneys, lungs, and heart tissues of each group of mice were dissected, and in vitro fluorescence imaging analysis was performed using a Maestro system (Caliper, IVIS Spectrum). The results are shown in Figure 7. Fluorescence recovery was observed in the tumors of mice that received AD(BHQ2)@PSSP-Cy3 injection after RT, indicating that the SeSe bonds in AD@PSSP can be broken under tumor radiotherapy. Furthermore, no significant fluorescence recovery was observed in the liver, kidneys, spleen, heart, and lungs of mice that received AD(BHQ2)@PSSP-Cy3 after RT, suggesting that the SeSe bonds in AD@PSSP do not break in these vital organs.

[0085] Example 6

[0086] The purpose of this embodiment is to verify in vivo whether AD-PEI can restore infection function after AD@PSSP is ruptured in SeSe.

[0087] Next, we further aim to confirm whether PEI still adheres to AD after SeSe cleavage and whether PEI maintains its basic function. Intravenous administration of different treatments to C57BL / 6 mice carrying TC-1 tumors, 24 hours after the TC-1 tumor was excised and immunofluorescence staining was performed. The specific scheme is as follows: according to the operation of Example 1, AD@PEG (using PEG to modify AD 11 ), AD@SSP (using SSP (SeSe-PEG) to modify AD 11 ), and AD@PSSP were prepared for use. 100 μl of normal saline, AD@PEG (1 × 10 8 pfu / ml), AD@SSP (1 × 10 8 pfu / ml), and AD@PSSP (1 × 10 8 pfu / ml) were intravenously injected into C57BL / 6 mice carrying TC-1 tumors (3 in each group). At the same time of intravenous injection, 2Gy of radiotherapy was given to the subcutaneous TC-1 tumors of each group of mice. 24h later, the TC-1 tumors of each group of mice were collected for quick frozen section using the Maestro system (Caliper, USA, IVIS spectrum). The section was stained with DAPI to show the nucleus, and examined by laser scanning confocal microscopy (3DHISTECH, Hungary, panorama MIDI, panorama 250 FLASH, panorama DESK). AD expresses GFP, and TC-1 cells infected with AD show GFP positive. The results are shown in Figure 8, and it can be seen that the RT+AD@PSSP group (G4) has the strongest GFP fluorescence, i.e. the tumor cells are infected with the most viral genes. In summary, AD@PSSP can effectively respond to ROS generated by RT to achieve the aggregation and targeted release of viruses at the tumor site, and at the same time, due to the timely shedding of the PEG shell, the infection ability of AD is effectively restored, and the presence of PEI further enhances the infectivity of AD.

[0088] Example 7

[0089] The purpose of this example is to verify the mechanism of AD-PEI enhancing DNA damage after RT.

[0090] RT mainly irradiates tumor by high-energy radiation, which acts on cancer cell DNA, causing damage and leading to cell death. AD-PEI not only can "dissolve" and infect tumor cells, but also can enhance the effect of RT by damaging cancer cell DNA. In this embodiment, HCT-116 cells (purchased from American Type Culture Collection (ATCC)) were treated with different reagents (PBS, PEI, AD and AD-PEI), and clonogenic assays were performed at different radiation doses (0 Gy, 3 Gy, 6 Gy and 9 Gy). The specific scheme is as follows: HCT-116 cells were seeded in 6-well plates at approximately 300 cells per well and treated with PBS, AD, PEI and AD-PEI (dose of 1 pfu / cell), respectively. Each treatment group was then subjected to different doses of X-ray radiation, with radiation doses of 0, 3, 6, 9 Gy, respectively. After 24 hours of radiation treatment, fresh culture medium was replaced, and cell growth was observed regularly. When the PBS-treated and non-irradiated group (0 Gy) formed obvious cell colonies, the culture was terminated. Then the culture medium was discarded, and each well of cells was washed with PBS three times, and then placed at room temperature for natural air drying. 1 mL of methanol was added to each well to fix the cells for 15 minutes, then the methanol was poured out, and the plate was air-dried again. The cells were stained with crystal violet solution for 15 minutes, then gently rinsed with tap water to remove excess stain, and then the plate was placed in the air to dry naturally. The number of colonies formed in each well of the 6-well plate was counted (each colony consisted of more than 50 cells) for analysis. The obtained data were fitted with a survival curve using a multi-target click model to calculate the biological parameters related to radiation. The formula for calculating the survival fraction (SF) is SF = 1-(1-e(-D / D0))N, where D0 represents the average 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. The results are shown in Figure 9, which shows that AD-PEI-treated HCT-116 cells have the highest radiation sensitivity, with a D0 of only 1.580 Gy, compared with 3.675 Gy for the PBS group and 2.732 Gy for the AD group.

[0091] This embodiment also explores the mechanism of AD-PEI enhancing the radiosensitivity of tumor cells. First, HCT-116 cells were subjected to multiple radiation treatments (6 Gy, 3 times), and then the cells were treated with PBS and AD-PEI, and proteomics analysis was performed. The specific scheme is as follows: the experimental raw data were obtained through the steps of protein extraction, quantification, quality control detection, restriction enzyme digestion and desalting, component separation and mass spectrometry analysis (this experiment was entrusted to Beijing Nuowozhengyuan Technology Co., Ltd.).

[0092] Subsequently, these data files were processed and analyzed using Proteome Discovserer version 2.2 software (Sequent HT, Thermo Fisher Scientific), and matched to the Uniprot protein database of Mus musculus (version date January 27, 2022, containing 86,515 sequences). By using AD... 11 The relative abundance of proteins enriched by PEI was calculated by dividing the normalized abundance value of each protein in the sample by the normalized 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 was used to rigorously screen the search results. Only peptide match with a confidence level of over 99% was considered a reliable peptide match, and only proteins containing at least one unique peptide were considered reliable proteins. During data processing, we only retained these validated reliable peptides and proteins and performed false discovery rate checks, excluding all peptides and proteins with a false discovery rate higher than 1%, thus ensuring the high quality and scientific validity of the experimental analysis results. The results showed that compared with the PBS group, the AD-PEI treatment group had increased expression of 195 proteins and decreased expression of 76 proteins. Figure 10 exemplarily shows 20 differentially expressed proteins, which have been reported in previous studies to enhance the RT effect.

[0093] Example 8

[0094] The purpose of this embodiment is to verify whether AD@PSSP can drain tumor antigens to lymph nodes after radiotherapy and whether it promotes antigen presentation.

[0095] Following tumor response (RT), the drainage of tumor antigens to lymph nodes is crucial for initiating an anti-tumor immune response. To determine whether AD-PEI could drain tumor antigens to lymph nodes in vivo, we seeded TC-1 cells into the dorsal aspect of the right thigh of C57BL / 6 mice. 100 μl of Cy5.5-labeled OVAs (Cy5.5-OVAs) (available from Acros) (1 mg / ml) was injected into the tissue surrounding the TC-1 tumor in C57BL / 6 mice. Subsequently, the mice were intravenously injected with 100 μl of saline and AD@SSP (10... 7 pfu / ml) or AD@PSSP(10) 7Simultaneously, 2 Gy of local tumor irradiation was administered, while the blank control group received no local tumor irradiation. Twenty-four hours later, drained lymph nodes were collected from the right side of the mice and analyzed using an in vitro fluorescence imaging system (Caliper, USA, IVIS Spectrum model). Flow cytometry (Accuri C6, BD, USA) was used to assess Cy5.5 positive cells and their antigen-presenting capacity in antigen-transmitting cells. The specific steps were as follows: First, the lymph nodes were crushed into a single-cell suspension using a 70 μm cell filter. After crushing, the cells were centrifuged at 1500 rpm for 5 minutes at room temperature to precipitate the cells, and the supernatant was discarded. Then, the cell pellet was resuspended in RPMI 1640 medium containing 2% fetal bovine serum, and the proportion of Cy5.5 positive cells in the lymph nodes was analyzed by flow cytometry (according to the instrument's operating instructions) to assess antigen uptake. Antigen presentation was measured using PE / Cy7-labeled anti-mouse H-2Kb (114616, clone name: 28-8-6) antibody bound to SIINFEKL.

[0096] As shown in Figure 11, the strongest Cy5.5 fluorescence signal was detected in the lymph nodes of the RT+AD@PSSP group. Figures 12A and B show the results of flow cytometry analysis of lymph node cells. The RT+AD@PSSP group contained the most Cy5.5 positive cells (A) and antigen-presenting cells for antibody presentation (B). This further demonstrates that AD-PEI can effectively drain tumor antigens released by RT to the lymph nodes and present more antigens to T cells.

[0097] Example 9

[0098] The purpose of this embodiment is to verify the anti-tumor efficacy, anti-tumor mechanism, and safety of RT combined with AD@PSSP.

[0099] Considering the in vitro therapeutic effects of AD@PSSP and RT, this embodiment further evaluated its anti-tumor ability in a TC-1 tumor-bearing C57BL / 6 mouse model immunized against pre-existing AD. The specific experimental protocol is as follows: In constructing the tumor model, firstly, on day 0, 3.5 × 10⁻⁶ mg / L of the drug was injected subcutaneously into the back (primary tumor site) of 6- to 8-week-old female C57BL / 6 mice. 6 TC-1 tumor cells. The primary tumor was treated with various injections of drugs on days 7, 9, 11, and 13 (including saline, AD, and AD@PP (using PP (PEI-PEG) modified AD)). 11 (Prepared according to the procedure in Example 1), AD@SSP and AD@PSSP, both at a dose of 5 × 10⁻⁶. 7pfu). The RT cycle was 8 days (from day 7 to day 14), 1 dose per day, 8 doses in total, 2 Gy per dose. On day 21, the primary tumor, distant tumor, spleen, and serum of the mice were analyzed. During the administration, the tumor volume and body weight were measured every two days. The formula for V is: V = 1 / 2ab 2 where "a" is the long axis of the tumor and "b" is the short axis of the tumor.

[0100] The results are shown in FIG. 13. In the groups without RT (G1-G4), the groups with PEG components (G3 and G4) were observed to have enhanced therapeutic effects compared to AD, highlighting the role of PEG in avoiding neutralizing antibodies and extending the circulation time of the drug. The AD@PSSP treatment group (G4) achieved the best therapeutic effect, which can be attributed to the inherent ROS in the tumor promoting the responsive release of AD@PSSP. In the treatment groups that received RT, the mice that used AD@PSSP in combination (G9) exhibited the most significant synergistic effect.

[0101] To further analyze the anti-tumor mechanism, the tumor samples after treatment were also analyzed. As shown in FIG. 14 A and B, the accumulation of OVs in the tumor was most significant in the RT+AD@PSSP treatment group. The proportion of cytotoxic T lymphocytes (CD3 + CD8 + cells) (CTLs) was also the highest in the RT+AD@PSSP group. The above results indicate that the efficacy of the RT+AD@PSSP group is mainly mediated by viral infection and antigen adaptive immune killing.

[0102] After the end of treatment, the lungs, liver, heart, kidneys, and spleen of the mice were removed for hematoxylin-eosin staining. The specific experimental steps are as follows: paraffin sections of the organs were prepared. Then the sections were sequentially placed in xylene I 10 min-xylene II 10 min-anhydrous ethanol I 5 min-anhydrous ethanol II 5 min-95% alcohol 5 min-90% alcohol 5 min-80% alcohol 5 min-70% alcohol 5 min-distilled water. Then the sections were placed in Harris hematoxylin for 3-8 min, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, and returned to blue with 0.6% ammonia water, and rinsed with running water. The sections were then placed in eosin staining solution for 1-3 min. Finally, the sections were sequentially dehydrated and transparentized in 95% alcohol I 5 min-95% alcohol II 5 min-anhydrous ethanol I 5 min-anhydrous ethanol II 5 min-xylene I 5 min-xylene II 5 min, and the sections were taken out of the xylene and slightly dried, and neutral balsam was used for mounting. Microscopic examination and image acquisition and analysis were performed. The results are shown in FIG. 15. No pathological damage was found in the main organs during the entire treatment process.

[0103] Example 10

[0104] This example aims to verify the anti-tumor effect of RT combined with AD@PSSP in an immunodeficient nude mouse model.

[0105] We further verified the synergistic therapeutic effect of AD@PSSP and RT in the absence of immunity in an immunodeficient mouse model (BALB / c nude mice) (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). The specific scheme is as follows: in the immunodeficient BALB / c nude mice, 1 × 10 8 HCT-116 cells were subcutaneously inoculated on day 0. On days 12 and 14, the corresponding drugs (including normal saline, AD@SSP (5 × 10 7 pfu), and AD@PSSP (5 × 10 7 pfu)) were administered. RT was performed from day 12 to day 15, once a day for 4 consecutive days, with a dose of 2 Gy each time. A blank control was set without RT treatment. The tumors were collected on day 21. The results are shown in Figures 16A and 16B, which show that the RT + AD@PSSP group showed a more obvious effect in inhibiting tumor growth compared to the other groups.

[0106] Example 11

[0107] This example aims to verify whether RT combined with AD@PSSP treatment can induce long-term immune memory.

[0108] Postoperative tumor recurrence is a major challenge in the clinical treatment of tumors. Therefore, RT is usually used as an adjuvant therapy after surgery to eliminate any residual potentially malignant cells and reduce the risk of local recurrence or distant metastasis. This example further evaluates the potential of AD@PSSP as an adjuvant therapy for postoperative RT, especially in inducing long-term immune memory. The evaluation method is as follows: 3.5 × 10 6 TC-1-Luc cells were subcutaneously inoculated in the right dorsal of C57BL / 6 mice to establish a primary tumor model on day 20. Three days later, the tumor was surgically removed, leaving 10% of the residual tumor. Then, different drugs (including normal saline and AD@PSSP (5 × 10 7 pfu)) were administered intravenously 4 times on days 0, 2, 4, and 6. RT was performed daily from day 0 to day 7, with 8 times of 2 Gy each time. A blank control was set without RT treatment. The mice were subjected to in vivo imaging before surgery, after surgery, and on days 20, 40, and 60 after surgery. The in vivo imaging analysis method is as follows: the mice were anesthetized with isoflurane, and then injected with luciferin (150 mg / kg) through the tail vein. After 10 min, the mice were imaged using an IVIS Spectrum imaging system (Xenogen, USA) for 5 min. The imaging data were analyzed using Living Image software (Xenogen, USA). 2+ and Ca 2+) to make 15 mg / mL fluorescein working solution, which was filtered through a 0.2 μm filter to remove bacteria. The injection amount was calculated at 10 μL / g body weight. Ten to fifteen minutes after intraperitoneal injection, the mice were placed on the imaging machine for analysis. The growth of the primary tumor volume was measured every other day until the 60th day. The results are shown in Figure 17, which shows that 80% of the C57BL / 6 mice receiving RT+AD@PSSP treatment survived, while the survival rate of the C57BL / 6 mice receiving postoperative RT alone was only 30%. On the 60th day, the C57BL / 6 mice in the RT+AD@PSSP group developed a distant tumor on the left side of the back inoculated with 3.5 x 10 6 em + + + -

[0109] On the 80th day, we evaluated the immune memory cells in the spleen cells and blood of the surviving mice by flow cytometry. The results are shown in Figure 19, which shows that the C57BL / 6 mice receiving RT+AD@PSSP treatment had a higher proportion of immune memory T cells T em (CD3 + CD8 + CD44 + CD62L - ) in the spleen cells and blood compared with the control group.

[0110] The cells of this example are a non-small cell lung cancer (TC-1-Luc) cancer cell line modified to express the human CD46 receptor and the Luc gene (donated by Beijing Hammer Biological Technology Co., Ltd.).

[0111] The examples described herein are illustrative and various modifications or changes in light thereof will occur to those skilled in the art, which should be included in the scope of the patent application.

[0112] Industrial Applicability

[0113] The present application provides a intravenous injection type oncolytic virus capable of enhancing the efficacy of radiotherapy, a preparation method and application thereof. The intravenous injection type oncolytic virus exhibits great potential in cancer treatment by combining radiotherapy and immunotherapy strategies, and is suitable for industrial application.

Claims

1. A intravenous injection type oncolytic virus which can reinforce the effect of radiotherapy, characterized in that, The intravenous injection type oncolytic virus capable of enhancing the radiotherapy effect is a genetically engineered oncolytic adenovirus AD 11 wherein the group is polyethyleneimine-diselenoic acid-polyethylene glycol (PEI-SeSe-PEG).

2. The radiotherapy potentiated oncolytic virus for intravenous injection according to claim 1, characterized in that, The intravenous oncolytic virus has a particle size of 133-138 nm.

3. The radiotherapy potentiated oncolytic virus for intravenous injection according to claim 1, characterized in that, The intravenous oncolytic virus has a dispersion index PDI of 0.21-0.

25.

4. The method of claim 1, wherein the method of preparing the intravenous injection type oncolytic virus capable of intensifying the radiotherapy effect is characterized by, The preparation method comprises the following steps: incubating an oncolytic adenovirus (AD 11 ) with an excess of PEI-SeSe-PEG at room temperature, and then removing unreacted active groups through an ultrafiltration tube, and after resuspension in PBS in the ultrafiltration tube, the intravenous oncolytic virus capable of enhancing radiotherapy efficacy is obtained.

5. The preparation method according to claim 4, characterized in that, The preparation method comprises: mixing PEI-SeSe-PEG solution with AD 11 , and then incubating in a room temperature environment to promote sufficient combination between them; then, removing unreacted active groups through an ultrafiltration tube; wherein, the ratio of the number of solutes to the number of virus particles is PEI-SeSe-PEG:AD 11 = 1:1.6×10 6 / Vp or above. 11 The preparation method comprises: mixing PEI-SeSe-PEG solution with AD 6. The preparation method according to claim 4, characterized in that, The PEG in the PEI-SeSe-PEG has a molecular weight of 20Kda or above.

7. The preparation method according to claim 4, characterized in that, In the ultrafiltration tube, the low-speed centrifugal revolution is 5000-10000 rpm, and the centrifugation time is 5-10 min; and / or The ultrafiltration tube has a molecular weight cut-off of 50-100 kDa.

8. The preparation method according to claim 4, characterized in that, The incubation is: mixing the aqueous solution containing PEI-SeSe-PEG with AD 11 After mixing uniformly, ultrasonic treatment is carried out at room temperature; wherein, the ultrasonic time is 30-60 min, and the room temperature is 20-25℃. Optionally, the concentration of PEI-SeSe-PEG in the aqueous solution containing PEI-SeSe-PEG is 1-10 mg / ml.

9. The intravenous oncolytic virus capable of enhancing the effect of radiotherapy for use in treating a malignant solid tumor disease according to claim 1.

10. A pharmaceutical composition comprising the intravenous oncolytic virus capable of enhancing the effect of radiotherapy according to claim 1 and a pharmaceutically acceptable excipient.

11. The pharmaceutical composition according to claim 10 for use in treating a malignant solid tumor disease.

12. A combination therapy, characterized in that, The combined treatment method comprises administering the intravenous oncolytic virus capable of enhancing the effect of radiotherapy according to claim 1 to a subject before, simultaneously with or after administering RT to the subject.

13. The method of combination therapy according to claim 12, wherein, The single dose of the RT is 2 Gy or more.

14. The method of combination therapy according to claim 12, wherein, The administration dose of the intravenous injection type oncolytic virus capable of enhancing the radiotherapy effect is (1×10 7 pfu-5×10 7 pfu).

15. The method of combination therapy according to claim 12, wherein, The subject has a malignant solid tumor disease, including but not limited to colon cancer, lung cancer, glioma.

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