Oncolytic virus preparation capable of crossing blood-brain barrier, preparation method therefor and use thereof

Abstract

Provided are an oncolytic virus preparation capable of crossing a blood-brain barrier, a preparation method therefor and a use thereof. The oncolytic virus preparation is an oncolytic virus modified by a polypeptide having a function of crossing a blood-brain barrier. The polypeptide having the function of crossing the blood-brain barrier is RVG29-Cys. The oncolytic virus is a genetically engineered oncolytic virus OH2 in which neurotoxic and immunosuppressive genes in a wild-type virus genome are knocked out and an immune-enhancing human granulocyte-macrophage colony stimulating factor gene is inserted. An OH2-PEG-RVG nanosystem is constructed by conjugating NHS-PEG-Mal to OH2, and then coupling a Mal group on NHS-PEG-Mal to a Cys group on RVG29-Cys, thus increasing the aqueous solubility of a drug while facilitating the therapeutic drug crossing of the blood-brain barrier to treat brain tumors, and preventing neutralizing antibodies and macrophages in vivo from neutralizing and phagocytosing the oncolytic virus, thereby prolonging the circulation time in vivo.

Description

Oncolytic virus formulation capable of crossing the blood-brain barrier, and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of nanomedicine, in particular to an oncolytic virus formulation capable of crossing the blood-brain barrier, and preparation method and application thereof. BACKGROUND

[0002] Oncolytic virotherapy is an important branch of tumor immunotherapy. Oncolytic virotherapy uses viruses with replication ability to selectively replicate and kill cancer cells. Oncolytic viruses (OVs) cause cancer cell death through different mechanisms, including apoptosis, pyroptosis and necroptosis. Oncolytic viruses release a wide range of tumor-associated antigens (TAA) / neoantigens or danger- and virus pathogen-related molecular patterns through direct oncolysis, thereby triggering inflammatory immune responses in the tumor microenvironment (TME). The highly immunosuppressive TME is a feature of malignant tumors, which is caused by the lack of tolerance of T cells to tumor-specific antigens and the development of systemic immunosuppression due to the isolation of T cells in the bone marrow. Oncolytic viruses can increase immune cell infiltration and trigger inflammation within the TME, which is crucial for breaking immune tolerance and can improve the responsiveness of tumors to immune checkpoint inhibitors. At the preclinical and clinical levels of malignant tumors, more and more oncolytic viruses are being widely tested, some of which are powerful oncolytic virus candidates such as adenovirus (DNX-2401), poliovirus (PVS-RIPO) and retroviral vector (Toca 511), etc., some of which have been marketed in the United States, China, etc.

[0003] Oncolytic viruses are currently available for the treatment of advanced melanoma, lung cancer, gastrointestinal tumors, etc., and are mainly used for patients who are resistant to second-line or even third-line treatment regimens. These patients often have tumors with refractory characteristics such as peripheral organ invasion, distant metastasis, and treatment resistance. In clinical practice, these patients are often accompanied by brain metastasis, among which lung cancer, melanoma and gastric cancer are more likely to metastasize to the intracranial space in the early stage. Brain tumors have a short course and the disease progresses rapidly after onset. If tumor hemorrhage and necrosis occur, the disease will suddenly worsen, and it can also present as a stroke-like onset. Currently, there are limited treatment options for brain tumors, mainly due to the presence of the blood-brain barrier (BBB), which is a complex protective barrier between the central nervous system and peripheral blood circulation. It is a tight structure composed of brain capillary endothelial cells (BCEC), astrocyte terminal feet, peripheral cells, and vascular basement membrane. Compared with other tissues, the blood-brain barrier has almost no intercellular space, greatly limiting the extracellular diffusion of solutes or drugs. In general, more than 98% of small molecule drugs and almost all macromolecular drugs cannot penetrate the blood-brain barrier. Brain-targeted drug delivery systems aim to address these issues, and they need to cross the blood-brain barrier and selectively increase drug concentrations in brain tissue. So far, drug delivery systems in the brain are mainly divided into three types: (1) absorption-mediated transcytosis (AMT); (2) carrier-mediated transport (CMT) system; (3) receptor-mediated transcytosis (RMT). Due to the overexpression of many different receptors by BCEC, RMT has become one of the most commonly used strategies for brain-targeted drug delivery. As expected, RMT has proven to enhance the BBB penetration of chemotherapeutic drugs and improve the treatment of brain tumors. As a selective active transport method, RMT has been widely used in brain-targeted research.

[0004] Nicotinic acetylcholine receptors (nAChR) are ion-gated channel receptors that are mainly expressed in neuronal cells. In addition, there are many studies on the expression of nAChR in brain endothelial cells and other tumor cells. Therefore, targeting nAChR can facilitate drug penetration through the BBB and achieve tumor-specific accumulation based on the RMT effect. Rabies virus glycoprotein peptide (RVG) is a glycoprotein expressed on the surface of rabies virus with a 29-amino-acid sequence that can specifically bind to nAChR expressed in neuronal cells, allowing the virus to enter. In particular, the a7 subunit of nAChR is widely expressed in the brain, including capillary endothelial cells. Although the tight junctions formed by brain endothelial cells prevent many drugs from crossing the BBB, studies have shown that the RVG29 peptide can allow therapeutic drugs to cross the BBB and rapidly reach brain tumor sites by binding to nAChR. SUMMARY

[0005] In view of the limitation of current brain tumor treatment methods, especially the hindrance of blood brain barrier to treatment, the application provides a oncolytic virus preparation capable of crossing the blood brain barrier, and a preparation method and application thereof.

[0006] OH2-PEG-RVG is obtained by combining NHS-PEG-Mal with OH2, and then combining the Mal group on NHS-PEG-Mal with the Cys group on RVG29-Cys; the particle size of OH2-PEG-RVG is 193-199 nm, and the dispersion index PDI is 0.08-0.1.

[0007] To achieve the object of the application, the application adopts the following technical solutions:

[0008] The application provides a oncolytic virus preparation capable of crossing the blood brain barrier, wherein the oncolytic virus preparation is a oncolytic virus modified by a polypeptide capable of crossing the blood brain barrier.

[0009] Preferably, the polypeptide capable of crossing the blood brain barrier is RVG29-Cys.

[0010] The oncolytic virus is further modified by PEG.

[0011] Preferably, the oncolytic virus is a genetically engineered oncolytic virus OH2 in which a neurotoxicity gene and an immunosuppression gene in a wild-type virus genome are knocked out, and a human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene is inserted.

[0012] The oncolytic virus preparation is named OH2-PEG-RVG, which is obtained by combining NHS-PEG-Mal with OH2, and then combining the Mal group on NHS-PEG-Mal with the Cys group on RVG29-Cys; the particle size of OH2-PEG-RVG is 193-199 nm, and the dispersion index PDI is 0.08-0.1.

[0013] The application further provides a preparation method of the oncolytic virus preparation capable of crossing the blood brain barrier according to the application, which comprises modifying a oncolytic virus by using a polypeptide capable of crossing the blood brain barrier and an active group to obtain the oncolytic virus preparation capable of crossing the blood brain barrier.

[0014] Preferably, the active group can improve the water solubility of the drug, while avoiding neutralization of antibodies in vivo and neutralization and phagocytosis of oncolytic viruses by macrophages, prolonging the circulation time in vivo.

[0015] Preferably, the active group is NHS-PEG-Mal. The preparation method comprises: combining NHS-PEG-Mal with OH2 to obtain OH2-PEG-Mal; and then combining the Mal group on NHS-PEG-Mal with the Cys group on RVG29-Cys to construct OH2-PEG-RVG.

[0016] Preferably, the oncolytic virus preparation capable of crossing the blood-brain barrier is OH2-PEG-RVG. There are abundant amino groups on OH2, which can be combined with NHS ester; and there are abundant Mal groups on PEG, which can be combined with Cys groups.

[0017] Preferably, in the active group NHS-PEG-Mal, the molecular weight of PEG is 20Kda.

[0018] Preferably, the preparation method of the oncolytic virus preparation capable of crossing the blood-brain barrier according to the present application comprises the following steps: incubating OH2 with different concentrations of NHS-PEG-Mal at room temperature (20-25℃), and then removing the active groups that do not react by using filter membranes and ultrafiltration tubes, and using barium iodide to stain PEG to determine the optimal reaction preparation amount (the PEG modification density is 45%). Among them, according to the ratio of the number of solutes to the number of virus particles, OH2:NHS-PEG-Mal (1.6×10 2 / vp).

[0019] Preferably, in the ultrafiltration tube, the low-speed centrifugation revolution is 5000-10000rpm, and the centrifugation time is 5-10min;

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

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

[0022] Preferably, the oncolytic virus preparation capable of crossing the blood-brain barrier needs to be stored at-80℃ for standby use.

[0023] The particle size of the prepared OH2-PEG-RVG is 193-199nm, and the dispersion index PDI is 0.08-0.1. The particle size is relatively uniform (PDI<0.3), and the nanoparticles in this range are stable and have the effect of crossing the blood-brain barrier.

[0024] In the active group with the function of improving the water solubility of the drug, avoiding neutralizing antibodies and macrophages in vivo, neutralizing and phagocytizing the oncolytic virus, and prolonging the circulation time in vivo, the water hydration reaction needs to be carried out before incubation, and the specific process of the water hydration reaction is as follows:

[0025] The active group with the function of improving the water solubility of the drug, avoiding neutralizing antibodies and macrophages in vivo, neutralizing and phagocytizing the oncolytic virus, and prolonging the circulation time in vivo is mixed with PBS at a ratio of 1-10 mg / ml to carry out the water hydration reaction to obtain a solution, and the water hydration reaction is carried out at room temperature, and the water hydration reaction time is at least 30 min, to obtain an aqueous solution of the active group with the antigen capture function, and the aqueous solution is stored at 4 DEG C.

[0026] In the polypeptide with the function of crossing the blood-brain barrier, the water hydration reaction needs to be carried out before incubation, and the specific process of the water hydration reaction is as follows:

[0027] The polypeptide RVG29-Cys powder with the function of crossing the blood-brain barrier is mixed with PBS at a ratio of 1-10 mg / ml to carry out the water hydration reaction to obtain a solution, and the water hydration reaction is carried out at room temperature, and the water hydration reaction time is at least 30 min, to obtain an aqueous solution of the polypeptide with the function of crossing the blood-brain barrier, and the aqueous solution is stored at -20 DEG C.

[0028] The present application provides a pharmaceutical composition comprising the oncolytic virus preparation capable of crossing the blood-brain barrier to treat brain tumors according to the present application and a pharmaceutically acceptable excipient.

[0029] The present application provides the use of the oncolytic virus preparation capable of crossing the blood-brain barrier to treat brain tumors, which is the use of the oncolytic virus preparation capable of crossing the blood-brain barrier to treat brain tumors or the pharmaceutical composition according to the present application in treating brain tumors.

[0030] The present application provides the use of the oncolytic virus preparation capable of crossing the blood-brain barrier to treat brain tumors in the preparation of a drug for treating brain tumors.

[0031] The present application provides a method for treating brain tumors, which comprises administering an effective amount of the oncolytic virus preparation capable of crossing the blood-brain barrier to treat brain tumors or the pharmaceutical composition according to the present application to a subject in need.

[0032] The administration mode comprises intravenous injection.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] Oncolytic viruses are a new type of immunotherapy, which can be used to treat advanced melanoma, lung cancer, gastrointestinal tumors, etc. However, the treatment of brain tumors is limited, mainly due to the presence of the blood-brain barrier. Generally, more than 98% of small molecule drugs and almost all macromolecular drugs cannot penetrate the blood-brain barrier. The brain-targeted drug delivery system aims to solve these problems, which needs to cross the blood-brain barrier and selectively increase the drug concentration in the brain tissue. The PEG connected to the oncolytic virus can significantly improve the water solubility of the drug, while avoiding the neutralization and phagocytosis of the oncolytic virus by neutralizing antibodies and macrophages in the body, prolonging the circulation time in the body. And RVG29 can quickly transport the oncolytic virus preparation to the brain tumor site by binding to nAChR.

[0035] The present application aims to solve the problem that the oncolytic virus on the market cannot break through the blood-brain barrier, and proposes the concept of "oncolytic virus-polypeptide nanosystem" for the treatment of brain tumors. While helping the treatment drug to cross the blood-brain barrier to treat brain tumors, the water solubility of the drug is improved, and the neutralization and phagocytosis of the oncolytic virus by neutralizing antibodies and macrophages in the body are avoided, prolonging the circulation time in the body. The nanosystem constructed in the present application provides a promising platform for oncolytic virus treatment of brain metastasis of solid tumors. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1A is a schematic diagram of drug construction in Example 1;

[0037] Figure 1B is an electron microscope and particle size characterization of OH2 in Example 1, the scale is 200nm;

[0038] Figure 1C is an electron microscope and particle size characterization of OH2-PEG-RVG in Example 1, the scale is 200nm;

[0039] Figure 2 is the killing of CT26 cells by OH2 and OH2-PEG-RVG at titers of 10 pfu / cell, 1 pfu / cell and 0.1 pfu / cell in Example 2;

[0040] Figure 3 is the intracellularization of OH2(Cy5.5)-PEG and OH2(Cy5.5)-PEG-RVG with different inhibitors in Example 3;

[0041] Figure 4 is the penetration of PBS, OH2(Cy5.5), OH2(Cy5.5)-PEG, OH2(Cy5.5)-RVG and OH2(Cy5.5)-PEG-RVG after culture in the Transwell chamber in Example 4;

[0042] Figure 5A shows the in vivo circulation time of OH2(Cy5.5), OH2(Cy5.5)-PEG, OH2(Cy5.5)-RVG and OH2(Cy5.5)-PEG-RVG in Example 5.

[0043] Figure 5B shows the brain tissue enrichment of OH2(Cy5.5), OH2(Cy5.5)-PEG, OH2(Cy5.5)-RVG and OH2(Cy5.5)-PEG-RVG at 48h in Example 5.

[0044] Figure 6 shows the Evans blue dye enrichment in brain tissue in Example 6.

[0045] Figure 7A shows the center time ratio of mine site experiment in Example 7.

[0046] Figure 7B shows the passing time of balance beam experiment in Example 7.

[0047] Figure 7C shows the cognitive index of new object recognition experiment in Example 7.

[0048] Figure 7D shows the immobility time ratio of tail suspension experiment in Example 7.

[0049] Figure 8A shows the tumor photos of pre-immunized model mice after treatment in Example 8, with a ruler of 1cm.

[0050] Figure 8B shows the tumor weight of pre-immunized model mice after treatment in Example 8.

[0051] Figure 9 shows the cytotoxic T lymphocyte ratio of tumor of pre-immunized model mice after treatment in Example 8, quantified by flow cytometry. DETAILED DESCRIPTION

[0052] The "OH2" involved in the present application is a recombinant human GM-CSF oncolytic type II herpes simplex virus, which is a new oncolytic virus derived from wild-type HSV-2 strain HG52.

[0053] The "NHS-PEG-MAL" referred to in the present application is a maleimide-polyethylene glycol-activated ester, which is a bifunctional linking molecule commonly used in bioconjugation reactions, particularly in the labeling and linking of proteins, antibodies, and other biomolecules. This molecule combines three main components: N-hydroxysuccinimide (NHS), polyethylene glycol (PEG), and maleimide (MAL). N-hydroxysuccinimide (NHS): NHS is a commonly used activating group that typically reacts with ester or amide groups to form stable amide bonds. In NHS-PEG-MAL, the presence of NHS enables one end of the PEG chain to react with other molecules containing amino groups, such as lysine residues on proteins or antibodies, forming a stable linkage. Polyethylene glycol (PEG): PEG is a hydrophilic polymer commonly used to increase the water solubility of molecules, reduce immunogenicity, and improve stability. In NHS-PEG-MAL, the PEG chain serves as a bridge between the NHS and MAL components, providing a flexible linker arm to accommodate different bioconjugation needs. Maleimide (MAL): MAL is a highly reactive group that can undergo Michael addition with molecules containing thiols, such as cysteine residues, forming a stable covalent linkage. In NHS-PEG-MAL, the presence of MAL enables the other end of the PEG chain to link with molecules containing thiols, enabling the connection between two different biomolecules.

[0054] The "RVG29" (sometimes referred to as "RVG" in the present application) referred to in the present application is a cell-penetrating peptide composed of 29 amino acid residues, derived from the rabies virus glycoprotein, with neural affinity and neurotransmitter modulation, which can penetrate the blood-brain barrier and enter brain cells. The "RVG29-Cys" (sometimes referred to as "Cys-RVG29" in the present application) referred to in the present application is a cysteine Cys connected to "RVG29".

[0055] In the present application, the term "oncolytic virus formulation capable of penetrating the blood-brain barrier", "oncolytic virus capable of penetrating the blood-brain barrier", or "oncolytic virus formulation" refers to an oncolytic virus-polypeptide nanosystem, which is an oncolytic virus modified by a polypeptide with the function of penetrating the blood-brain barrier. In some embodiments, the "oncolytic virus formulation capable of penetrating the blood-brain barrier", "oncolytic virus capable of penetrating the blood-brain barrier", or "oncolytic virus formulation" may contain other substances brought about during the production process in addition to the active substance that performs the intended function, i.e., the oncolytic virus modified by a polypeptide with the function of penetrating the blood-brain barrier. In some embodiments, the oncolytic virus modified by a polypeptide with the function of penetrating the blood-brain barrier is further modified by an active group. In further embodiments, the active group is NHS-PEG-MAL.

[0056] Hereinafter, the present application will be described in detail with reference to the embodiments and the accompanying drawings. It should be noted that the following embodiments are merely illustrative, and the following embodiments do not limit the scope of protection of the present application.

[0057] Embodiments

[0058] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0059] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0060] In the following examples, OH2 is a gift from Wuhan Binhu Biotechnology Co., Ltd.

[0061] In the following examples, NHS-PEG 20k -Mal was purchased from Xi'an Ruishi Biological Technology Co., Ltd.

[0062] In the following examples, RVG29-Cys was purchased from Qiangyao Biological Technology Co., Ltd.

[0063] In the following examples, the culture medium of the mouse colon cancer (CT26) cell line is 1640 medium, and the conventional culture method is as follows: using 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, the cells are cultured in a humidified environment containing 5% carbon dioxide at 37°C.

[0064] In the following examples, the culture medium of the mouse brain microvascular endothelial cell (bEnd.3) cell line is DMEM, and the conventional culture method is as follows: using DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, the cells are cultured in a humidified environment containing 5% carbon dioxide at 37°C.

[0065] In the following examples, OH2(Cy5.5) is prepared by incubating OH2 and Cy5.5-NHS at a ratio of 50:1 at 4°C for 12 hours, and removing unreacted Cy5.5-NHS by ultracentrifugation at 1500g and 4°C for 10 minutes.

[0066] Example 1 Construction of OH2-PEG-RVG

[0067] The oncolytic virus OH2-PEG-RVG capable of crossing the blood-brain barrier is constructed by the following method:

[0068] (1) 540 μL of NHS-PEG 20k -Mal solution (10 mg / mL) and 100 μL of OH2 (1 × 10 7The excess NHS-PEG was removed by ultracentrifugation at 1500g for 5-10 min at 4°C 20k -Mal, to obtain OH2-PEG-Mal (or OH2-PEG). Excess RVG29-Cys (100 μL, 10 mg / mL RVG29-Cys) was added to the mixture, and the mixture was incubated at 4°C for 12 h. The excess RVG29-Cys was removed by ultracentrifugation at 1500g for 5-10 min at 4°C;

[0069] (2) The solution obtained in (1) was further purified by ultrafiltration using a 100 kDa ultrafiltration tube. The residue in the filter membrane was dissolved with PBS, and the obtained solution was the OH2-PEG-RVG solution, which was stored at -80°C for later use;

[0070] (3) The morphology and particle size of OH2 and OH2-PEG-RVG were characterized by transmission electron microscopy (FEI, Tecnai G2 20S-TWIN, 200 kV) and a laser particle size analyzer (Malvern, Zetasizer Nano ZS90).

[0071] The schematic diagram of the method is shown in FIG. 1A, and the electron microscopy and particle size characterization results of OH2 and OH2-PEG-RVG are shown in FIGS. 1B and 1C. FIG. 1B is a transmission electron micrograph of OH2, with a particle size of about 155.7 nm and a dispersion index (PDI) of 0.2563. FIG. 1C is a transmission electron micrograph of the prepared OH2-PEG-RVG, which is spherical and has a particle size of about 193 nm and a dispersion index (PDI) of 0.0832. Compared with OH2, the particle size increases by about 40 nm, and the change in electron microscopy morphology and the increase in particle size indicate that OH2-PEG-RVG is successfully prepared, which is OH2 modified by PEG and RVG.

[0072] Example 2 verifies the killing function of OH2-PEG-RVG on tumors

[0073] In this example, a mouse colon cancer (CT26) cell line (purchased from Beijing Yanyong Biological Technology Development Co., Ltd.) was used as the research object to verify the killing function of OH2-PEG-RVG on tumors, and the method was as follows:

[0074] (1) CT26 cells in the logarithmic growth phase in the incubator were taken, and the cell suspension concentration was adjusted to 1×10 5Cells were suspended at 100 μl per well in a 96-well cell culture plate, resulting in a cell density of 1 × 10⁶ cells / ml in each well. 4 There are 27 experimental wells in total.

[0075] (2) Place the cells in a humidified environment containing 5% carbon dioxide at 37°C for 14-16 hours, when the cells are completely attached to the wall but have not yet proliferated, and then remove the culture plate.

[0076] (3) Divide the 27 wells into groups A, B, and C, with 9 wells in each group. Add 10 μl of PBS to the first 3 wells of group A, and add 1×10 μl of PBS to the middle 3 wells. 7 10 μl of OH₂ at pfu / ml is equivalent to 10 pfu / cell per well. Add 1×10⁻⁶ pfu / ml to the last 3 wells. 7 10 μl of OH2-PEG-RVG at pfu / ml is equivalent to 10 pfu / cell per well.

[0077] Add 10 μl of PBS to the first 3 wells of group B, and add 1×10⁻⁶ PBS to the middle 3 wells. 6 10 μl of OH₂ at pfu / ml is equivalent to 1 pfu / cell per well. Add 1 × 10⁻⁶ pfu / ml to the last 3 wells. 6 10 μl of OH2-PEG-RVG at pfu / ml is equivalent to 1 pfu / cell per well.

[0078] Add 10 μl of PBS to the first 3 wells of group C, and add 1×10⁻⁶ PBS to the middle 3 wells. 5 10 μl of OH₂ at pfu / ml is equivalent to 0.1 pfu / cell per well. For the last three wells, add 1×10⁻⁶ pfu / ml. 5 10 μl of OH2-PEG-RVG at pfu / ml is equivalent to 0.1 pfu / cell per well.

[0079] (4) After culturing the cells for another 3 days, remove the culture plate and add 100 μl of CCK8 mixture (the CCK8 mixture is a 1:10 mixture of CCK8 and 1640 medium, where CCK8 was purchased from Beijing Lanbolide Trading Co., Ltd.) to each well. Incubate at 37℃ for 0.5 h. The light absorbance measured by CCK8 was measured at 450 nm. Cells cultured only in the medium were used as positive controls (OD positive control), and the medium was used as negative controls (OD negative control). Cell viability was calculated as follows: Cell viability (%) = (OD experiment - OD negative control) / (OD positive control - OD negative control) × 100.

[0080] The results are shown in Figure 2. It can be seen that, under the same dosage (10 pfu / cell, 1 pfu / cell and 0.1 pfu / cell), OH2-PEG-RVG killed CT26 cells with the same efficiency as OH2, and showed a dose-dependent effect, i.e. the higher the dosage of OH2-PEG-RVG used, the stronger the killing ability of OH2-PEG-RVG on CT26 cells. For example, when the dosage of OH2-PEG-RVG used was 10 pfu / cell, 1 pfu / cell and 0.1 pfu / cell, the activity of CT26 cells was about 10%, 50% and 80%, respectively.

[0081] Example 3 verifies the internalization of OH2-PEG-RVG into the brain

[0082] The cells used in this example were a mouse brain microvascular endothelial cell (bEnd.3) cell line (purchased from Beijing Yenan Biotechnology Development Co., Ltd.).

[0083] (1) Take the bEnd.3 cells in the logarithmic growth phase in the incubator, use DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, adjust the cell suspension concentration to 2 x 10 6 cell / ml, and plate 100 μl of cell suspension in each well of a 48-well cell culture plate. At this time, the cell density in each well of the 48-well plate to be tested is 2 x 10 5 cell / well, and a total of 42 experimental wells are set up.

[0084] (2) Place the cells in a 37°C humidified environment containing 5% carbon dioxide for 14-16 h, and take out the culture plate when the cells are completely adherent but have not yet proliferated.

[0085] (3) Divide the 42 experimental wells into groups A and B, 21 wells in each group. In group A, add 10 μL of room temperature PBS, 10 μL of PBS at 4°C, 10 μL of 0.45 M sucrose, 10 μL of 30 μM chlorpromazine, 10 μL of 100 μM EIPA (5-(N-ethyl-N-isopropyl) amiloride), 10 μL of 30 μM nystatin and 10 μL of 1 mg / mL ammonium chloride, etc. to each set of 3 wells, respectively. At the same time, add 10 μL of OH2(Cy5.5)-PEG to each well in group A. Group B is the same as group A except that OH2(Cy5.5)-PEG-RVG is added instead of OH2(Cy5.5)-PEG.

[0086] (4) After 12 h of conventional cell culture, detect the Cy5.5 fluorescence of the cells by flow cytometry. According to the cell grouping circle gate in the flow cytometry, determine the relative cell uptake amount (%).

[0087] The results are shown in Figure 3, which shows that the uptake of both nanoparticles by bEnd.3 at 4°C was significantly reduced (more than 90%) compared with 37°C, a fact that implies that the internalization of these particles is mainly energy-dependent. Both sucrose and chloφromazine, two inhibitors of clathrin-coated vesicle formation, significantly reduced the cellular uptake of nanoparticles by more than 30% compared with other specific inhibitors. This indicates that the main pathway of internalization of OH2-PEG and OH2-PEG-RVG is clathrin-mediated endocytosis. However, when the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and the microcyst-mediated endocytosis inhibitor nystatin were used, there was little effect on the uptake of nanoparticles, indicating that these pathways were not seriously affected. In addition, the inventors used ammonium chloride, a lysosome-promoting drug, to verify the function of endosomes / lysosomes in cellular uptake. For OH2(Cy5.5)-PEG and OH2(Cy5.5)-PEG-RVG, the amount of uptake was reduced by more than 50%, indicating that these compartments are involved in the uptake of nanoparticles. In addition, lysosomes are expected to be involved in drug release and particle destruction, while endosome transport can be necessary for the passage of intact particles through the cell barrier. It can thus be seen that the internalization of OH2-PEG-RVG mainly occurs through energy-dependent endocytosis rather than an energy-independent mechanism.

[0088] Example 3 verifies the ability of OH2-PEG-RVG to cross the blood-brain barrier in vitro

[0089] (1) Using a 24-well plate with a transwell chamber, CT26-LUC cells (purchased from Beijing Yenan Biotechnology Development Co., Ltd.) were cultured in the bottom layer of the 24-well plate using 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and the density of CT26-LUC cells in the bottom layer was 1 x 10 4 cells / well, and bEnd.3 cells were cultured in the chamber, and the density of cells in the chamber was 3 x 10 3 cells / well, and a total of 20 experimental holes were set. The cells were cultured in a humidified environment containing 5% carbon dioxide at 37°C.

[0090] (2) After the cells were routinely cultured for 7 days, the cells reached confluence, and the TEER of the monolayer cells in the chamber was measured using a Millicell ERS volt-ohm meter (USA, Millipore Co.), and monolayer cells with a TEER of at least 200 Ω·cm 2 were selected for further study.

[0091] (3) Add OH2(Cy5.5), OH2(Cy5.5)-PEG, OH2(Cy5.5)-RVG or OH2(Cy5.5)-PEG-RVG into the upper chamber for 12h (the amount of drug added is equivalent to the amount of OH2 1 μL). At the same time, use PBS instead of the medium in the upper chamber.

[0092] (4) Image the fluorescence in the bottom layer of the 24-well plate and the bEnd.3 (transwell chamber) layer using IVIS Spectrum (USA). The results are shown in Figure 4, and the drug added in the upper chamber permeates the brain vascular endothelial cells at the bottom of the chamber to the lower layer, proving that OH2-PEG-RVG successfully crosses the blood-brain barrier in vitro.

[0093] Example 4 verifies the circulation time and in vivo distribution of OH2-PEG-RVG in vivo

[0094] (1) Administer OH2(Cy5.5)-PEG, OH2(Cy5.5)-RVG and OH2(Cy5.5)-PEG-RVG (equivalent to 10 6 pfu / kg (mouse weight) of OH2) and OH2(Cy5.5) (10 6 pfu / kg (mouse weight)) intravenously to female BALB / C mice (6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) (17±3 g mouse weight). There are 3 mice in each group.

[0095] (2) At 2, 6, 12, 24 and 48h after administration, extract 10 μL of blood sample from the tail vein and place it in a heparinized microcentrifuge tube, and use IVIS Spectrum fluorescence imaging to detect the blood sample.

[0096] (3) Collect the mouse brain at 48h. Image the mouse brain sample using IVIS Spectrum (USA). The results are shown in Figure 5, proving that OH2-PEG-RVG prolongs the in vivo circulation time (Figure 5A) and increases brain enrichment (Figure 5B) compared to OH2.

[0097] Example 5 verifies the damage of OH2-PEG-RVG to the blood-brain barrier

[0098] Administer OH2(Cy5.5), OH2(Cy5.5)-PEG, OH2(Cy5.5)-RVG or OH2(Cy5.5)-PEG-RVG (all equivalent to 10 6pfu / kg (mouse body weight) OH2) with 3 mice per group. On the third day after the last injection, Evans blue (3%; 4 mL / kg mouse body weight) was infused slowly through the tail vein, and euthanasia was performed 2 h later. Afterwards, about 200 μΐ of PBS was injected into the mice to eliminate the residual dye. The mice brains were dissected and each hemisphere was homogenized in 1 mL of formamide at 55 °C for 24 h. The supernatant was collected after centrifugation at 12000 g for 20 min, and the absorbance at 620 nm was used to quantify the concentration of Evans blue. The results are shown in Figure 6, which shows no significant difference between OH2-PEG-RVG and OH2, demonstrating that OH2-PEG-RVG did not compromise the integrity of the blood-brain barrier.

[0099] Example 6 verifies the effect of OH2-PEG-RVG on the behavior of mice

[0100] On days 0, 2, and 4, PBS and OH2-PEG-RVG (equivalent to 10 6 pfu / kg OH2) were injected intravenously into female C57BL / 6 mice (6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) (17 ± 3 g mouse body weight) with 3 mice per group.

[0101] The open field test was performed on day 7 to assess locomotor activity and anxiety-like behavior, using a simple 44 cm x 44 cm x 30 cm open field arena. The activity of the animals in the open field for 10 minutes was recorded by a video capture or camera system. The results were analyzed using LimeLight 5 software.

[0102] The balance beam test was performed on days 10-12. On the first day of the balance beam test, the mice were trained to pass through a square balance beam into a small dark box, with each mouse trained three times, with a 2-hour interval between each training. On the second day, the steps of the first day were repeated. The third day was the test phase, in which it was allowed to pass through the balance beam to the small box. The time required to reach the box and the number of paper strips were recorded. If it did not reach the small box, the time was recorded as 60 seconds.

[0103] The novel object recognition test was performed on day 15, which was performed in an open field (44 cm x 44 cm x 30 cm). Two identical objects (A and B, ensuring that the objects had no odor, were fixed and could not move) were placed in the device, with the objects about 5-10 cm away from the wall on both sides. The mouse was placed in the device with its back towards the objects and at an equal distance from the objects, and after 5 minutes, the mouse was placed back in the cage. After 1 hour, one of the two identical objects was replaced with another object and placed in the device (AC or BC). Similarly, the mouse was placed in the device with its back towards the objects and at an equal distance from the objects for 5 minutes. The exploration time of the mouse on each object was recorded using a camera device and software LimeLight 5. The calculation formula of the cognitive index (RI) is: RI = new object / (new object + old object) x 100%.

[0104] The tail suspension test was performed on day 18, and the mouse tail was fixed about 1 cm from the magnet with tape, with its head hanging down. At the same time, it is important to ensure that all parts of the mouse body, except the tail and the instrument, do not touch the tail suspension device during the suspension process. The test lasted for 6 minutes, and the percentage of still time of the animal in the last 4 minutes was analyzed using Freeframe 5 software.

[0105] The results are shown in FIG. 7A (mine field experiment), FIG. 7B (balance beam experiment), FIG. 7C (novel object recognition experiment), and FIG. 7D (tail suspension experiment), and it can be seen that in the mine field experiment, balance beam experiment, novel object recognition experiment, and tail suspension experiment, there was no significant difference between the PBS injection group of mice and the OH2-PEG-RVG injection group of mice in each key parameter, indicating that OH2-PEG-RVG had no significant effect on the behavior, psychology, and memory function of mice.

[0106] Example 7 verifies the anti-tumor efficacy of OH2-PEG-RVG in mice with existing anti-virus antibodies

[0107] In China, most people are immunocompromised in childhood, infected with herpes simplex virus, and have developed neutralizing antibodies against herpes simplex virus. When these people receive oncolytic virus treatment based on herpes simplex virus, the pre-existing immunity can eliminate the oncolytic virus and affect the treatment efficiency. To explore this challenge, the present application constructed a mouse model with immunity to OH2 by pre-injecting the virus, i.e., subcutaneously injecting a low dose of OH2 (3 x 10 5 pfu) into female BALB / c mice (6-8 weeks old) on days -42, -35, and -14, and on day -7, the mouse brain was injected intracranially with 5 x 10 55 μl CT26-LUC cells and 5 μl Matrigel (needle insertion site: 1 mm right to the sagittal suture, 1 mm in front of the fontanel, needle insertion depth 3 mm). On day 0, 2 and 4, different drugs (PBS, OH2, OH2-PEG, OH2-RVG and OH2-PEG-RVG, dose 10 6 pfu). On day 9, the brain of the mouse was dissected, the brain metastases were obtained and weighed.

[0108] The experimental results show that OH2-PEG-RVG has the best curative effect, that is, the tumor shrinks the most, and shows the strongest anti-tumor ability to brain metastases (Fig. 8A, Fig. 8B).

[0109] Take 0.2 g of tumor tissue in the brain, cut the tumor tissue into small pieces in the digestive juice (digestive juice: trypsin diluted with 1640 medium at 1:20, the final digestive juice contains 0.5 mg / mL collagenase IV and 20 μg / mL DNase I), and digest for 30 min in a 37°C shaking bed to obtain a single cell suspension. The digested tumor cells are stained with CD3 and CD8, and then subjected to flow detection to evaluate the infiltration of immune cells. As shown in Fig. 9, the proportion of cytotoxic T lymphocytes (CD3+CD8+ cells) in the primary and distant tumors in the OH2-PEG-RVG group is the highest.

[0110] The examples described herein are used as illustrations only and various modifications or changes in light thereof will be obvious to those skilled in the art, which should be included in the scope of the patent application.

[0111] Industrial applicability

[0112] The present application provides a kind of oncolytic virus preparation capable of crossing blood-brain barrier and its preparation method and application, the oncolytic virus preparation provided has prolonged in vivo circulation time, and can cross blood-brain barrier, can provide platform for the treatment of solid tumor brain metastasis, suitable for industrial application.

Claims

1. An oncolytic virus preparation capable of crossing the blood-brain barrier, characterized in that, The oncolytic virus preparation is an oncolytic virus modified with peptides that have the function of crossing the blood-brain barrier; The polypeptide with the ability to cross the blood-brain barrier is RVG29-Cys.

2. The oncolytic virus preparation according to claim 1, characterized in that, The oncolytic virus was also modified with PEG.

3. The oncolytic virus preparation according to claim 2, characterized in that, The oncolytic virus is a genetically engineered oncolytic virus OH2, which has the neurotoxic and immunosuppressive genes knocked out of the wild-type virus genome and has human granulocyte-macrophage colony-stimulating factor inserted to enhance immunity.

4. The oncolytic virus preparation according to claim 3, characterized in that, The oncolytic virus preparation is named OH2-PEG-RVG, which is obtained by combining NHS-PEG-Mal with OH2, and then combining the Mal group on NHS-PEG-Mal with the Cys group on RVG29-Cys; the particle size of OH2-PEG-RVG is 193-199 nm, and the dispersion index PDI is 0.08-0.

1.

5. A method for preparing an oncolytic virus preparation capable of crossing the blood-brain barrier according to any one of claims 1-4, characterized in that, The preparation method includes modifying the oncolytic virus with peptides and active groups that have the function of crossing the blood-brain barrier to obtain the oncolytic virus preparation that can cross the blood-brain barrier.

6. The preparation method according to claim 5, characterized in that, The active group is NHS-PEG-Mal.

7. The preparation method according to claim 5, characterized in that, The preparation method includes: By combining NHS-PEG-Mal with OH2, OH2-PEG-Mal is obtained; then, by combining the Mal group on NHS-PEG-Mal with the Cys group on RVG29-Cys, OH2-PEG-RVG is obtained.

8. The preparation method according to claim 7, characterized in that, OH2 was incubated with NHS-PEG-Mal at room temperature, and then unreacted active groups were removed by passing the mixture through a filter membrane and ultrafiltration tube. PEG was stained with barium iodide to determine the reaction dosage. The solute-to-virus particle ratio was OH2:NHS-PEG-Mal = 1.6 × 10⁻⁶. 2 / vp.

9. The preparation method according to claim 5, characterized in that, The active group undergoes a hydration reaction before incubation, and / or peptides with the ability to cross the blood-brain barrier undergo a hydration reaction before incubation.

10. A pharmaceutical composition comprising an oncolytic virus preparation capable of crossing the blood-brain barrier to treat brain tumors as described in any one of claims 1-4, and pharmaceutically acceptable excipients.

11. The use of any one of the oncolytic virus preparations capable of crossing the blood-brain barrier to treat brain tumors, or the pharmaceutical composition of claim 10, in the treatment of brain tumors.

12. A method for treating brain tumors, comprising administering an effective amount of an oncolytic virus preparation capable of crossing the blood-brain barrier to treat brain tumors, as described in any one of claims 1-4, or a pharmaceutical composition as described in claim 10, to a subject in need.

13. The method of claim 12, wherein the administration method includes intravenous injection.

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