Oncolytic virus formulation and preparation method thereof

Abstract

The present invention provides an oncolytic virus formulation and preparation method thereof. The oncolytic virus formulation comprises cell vesicles derived from apoptotic tumor cells and oncolytic viruses coated in the cell vesicles as an effective component. The oncolytic virus formulation uses cell vesicles derived from tumor cells themselves to coat the oncolytic viruses, so as to evade the body's immune system attack and can be targeted to the tumor treatment site, and improve the tumor-killing effect.

Claims

1. A method for preparing an oncolytic virus formulation, comprising mixing oncolytic viruses and tumor cells in a ratio of 1:1 to 20:1 so as to infect tumor cells, and culturing the infected tumor cells to induce apoptosis at 37 C. and 5% oxygen content, collecting microparticles released from the apoptotic tumor cells within 48-72 hours, wherein the microparticles are the oncolytic virus formulation generated after the oncolytic virus is coated with cell vesicles; wherein the collection of the microparticles released from the apoptotic tumor cells comprises removing the tumor cells and debris by an ultracentrifuge at a centrifugal force of not more than 5000 g, and collecting the microparticles by the ultracentrifuge at a centrifugal force of 10000 g.

2. The method according to claim 1, wherein the tumor cells include cells of ovarian cancer, breast cancer, lung cancer, gastric cancer, colon cancer, liver cancer, pancreatic cancer or prostate cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) This application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

(2) FIG. 1a is an electronic micrograph of microparticles released from cultured A549 human lung tumor cells after being treated with oncolytic adenovirus.

(3) FIG. 1b is a particle size distribution graph of microparticles generated from the apoptosis of oncolytic adenovirus-induced human lung cancer cells. In FIG. 1b, y-axis represents the number density of particles in a microparticle sample to be measured, the value of x-axis corresponding to the red curve peak represents the diameter range for most of the microparticles in the sample.

(4) FIG. 2a shows that microparticles generated after cultured tumor cells are treated with oncolytic adenoviruses, contain oncolytic adenovirus DNA.

(5) FIG. 2b shows that microparticles coating oncolytic adenoviruses. after tumor cells are treated with oncolytic adenoviruses,

(6) FIG. 3 shows graph of the effect of oncolytic adenovirus-coated microparticles killing variety of human tumor cells. In the graph, a, b, c and d represents A2780 human ovarian cancer cell line, MCF-7 human breast cancer cell line, A549 human lung cancer cell line and SNU1 human gastric cancer cell line respectively; g, h, i and j represents Caco2 human colon cancer cell line, HepG2 human hepatoma cell line, ASCP-1 human pancreatic cancer cell line, LINCap human prostate cancer cell line respectively.

(7) FIG. 4a and FIG. 4b show the content of alanine transaminase (ALT) and creatinine (CRE) in the serum of mouse in each group, which shows that the oncolytic adenovirus-coated microparticles have no obvious toxic side effects on the body.

(8) FIG. 5a shows the tumor volume of A549 tumor-bearing mouse after being treated with phosphate buffered saline (PBS), cell vesicles (MP), oncolytic adenoviruses (OA), and oncolytic adenovirus-coated microparticles (OA-MP) respectively on the 5th day and 21th day; FIG. 5b shows the tumor volume of A549 tumor-bearing mouse detected at different time points after the mouse is treated with PBS, MP, OA, OA-MP respectively; FIG. 5c shows the survival rate of A549 tumor-bearing mice after being treated with PBS, MP, OA respectively; FIG. 5d shows the growth of A2780 tumor in nude mice after being treated with PBS, MP, OA and OA-MP respectively.

DETAILED DESCRIPTION

(9) In order to confirm that tumor cell-derived cell vesicles are capable of coating oncolytic adenoviruses, effectively killing tumor cells and having no obvious toxic side effects in vivo, the present invention is further described as below with reference to drawings and the examples.

(10) Various tumor cells, oncolytic adenoviruses and test animals used in the following examples:

(11) A2780 human ovarian cancer cell line, MCF-7 human breast cancer cell line, A549 human lung cancer cell line, SNU1 human gastric cancer cell line, Caco2 human colon cancer cell line, HepG2 human hepatoma cell line, ASCP-1 human pancreatic cancer cell line, LINCap human prostate cancer cell lines, all are commercially available from American ATCC Center or from China Center for Type Culture Collection (CCTCC).

(12) Oncolytic adenoviruses are available from Shanghai Sanwei Biotechnology Co., Ltd., have the name of recombinant human type 5 adenovirus (H101). 200 female nude mice, each weighing 18 grams, and are purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences and Peking Union Medical College.

Example 1: Oncolytic Adenoviruses were Used to Induce Apoptosis of Human Lung Cancer Cells to Produce Microparticles

(13) 1. Experimental Steps

(14) A549 human lung cancer cells were cultured in DMEM cell culture fluid, so that the number of cells reached 110.sup.7; 110.sup.8 oncolytic adenoviruses were added to the culture fluid to infect human lung cancer cells; at 37 C. and 5% oxygen content, the infected tumor cells were cultured; at 48th hour after administration of oncolytic adenoviruses, when the tumor cells became smaller and dimmer, the supernatant of the human lung cancer cell culture fluid was centrifuged successively at the centrifugal force of 1000 g and 5000 g for each 10 minutes, the cells and debris were removed, and the supernatant after centrifugation was further centrifuged at the centrifugal force of 10000 g for 2 hours, the precipitate was collected to obtain microparticles generated from cell vesicles derived from apoptotic A549 human lung cancer cells coating oncolytic adenoviruses.

(15) 2. Experimental Results

(16) The microparticles prepared above were resuspended with 1 ml of 0.9% (g/ml) physiological saline. After fixed on glass, the microparticles were observed under a transmission electron microscope. (see FIG. 1a). At the same time, the particle sizes of the microparticles were detected by Mastersizer 2000 particle size analyzer and a particle size distribution result was obtained (see FIG. 1b), the particle size distribution for the most of the microparticles in a sample was in the range of 100-1000 nm.

Example 2: Cell Vesicles Generated from the Apoptosis of Oncolytic Adenovirus-Induced Human Lung Cancer Cells Coat Oncolytic Adenovirus DNA

(17) 1. Experimental Steps

(18) A549 human lung cancer cells were cultured, so that the number of cells reached 110.sup.7; 110.sup.8 oncolytic adenovirus particles were added to the culture fluid; at 48th hours after administration of oncolytic adenoviruses, when the tumor cells became smaller and dimmer, microparticles (generated after cell vesicles derived from apoptotic A549 human lung cancer cells coat oncolytic adenoviruses) generated from apoptotic A549 human lung cancer cells in the supernatant of the human lung cancer cell culture fluid was collected according to the steps of Example 1.

(19) On the one hand, the microparticles were treated with proteinase K, the membrane was lyzed and DNA molecules were separated, meanwhile oncolytic adenoviruses were treated and separated to prepare DNA molecules contained therein.

(20) On the other hand, the microparticles obtained above were stained with PKH67 (shown in green), while adenoviruses in the microparticles were stained with PE fluorescently-labeled anti-adenovirus antibody (shown in red), and they were observed under a confocal fluorescence microscope.

(21) As a contrast, the cell vesicles obtained from the above-mentioned A549 human lung cancer cells were stained with PKH67 (shown in green) and cell vesicles were stained with PE fluorescently-labeled anti-adenoviral antibody (shown in red), and they were observed under a confocal fluorescence microscopy.

(22) The preparation method of the above-mentioned cell vesicles comprised the following steps: culturing tumor cells, irradiating the cells with ultraviolet rays for 30 minutes, when the tumor cells significantly became smaller and dimmer at 48th hour after UV irradiation, the supernatant of the culture liquid of mouse hepatoma cells was subjected to gradual centrifugation, that is centrifuged in sequence at the centrifugal force of 500 g, 1000 g, and 5000 g for each 10 minutes, and then centrifuged at the centrifugal force of 14000 g for 1 minute to remove cells and debris, the centrifuged supernatant was further centrifuged at the centrifugal force of 14000 g for 1 hour and precipitation was collected to get cell vesicles.

(23) 2. Experimental Results

(24) Electrophoresis was carried out on DNA prepared above respectively, and the results of the electrophoresis were shown in FIG. 2a. In FIG. 2a, OA represented oncolytic adenovirus DNA, OA-MP represented DNA of microparticles which coat oncolytic adenoviruses, DNA molecular weight standard markers were at the leftmost lane. The oncolytic adenovirus genome is known to be about 23 kb in size, while the DNA electrophoresis marker contained in the microparticle sample had the same size with the known oncolytic adenovirus genome. At the same time, the results of fluorescence microscope observation were shown in FIG. 2b, PKH67 was used to stain and both MP group (cell vesicle group) and OA-MP group (microparticle group) showed green color; PE fluorescence label was used to stain anti-adenovirus antibody and MP group did not show red, and OA-MP group showed red; from the pictures provided after overlapping the stained pictures of two groups, it could be seen that in the OA-MP group green and red colocalization, indicating that the microparticles of the present invention coat oncolytic adenoviruses.

Example 3

(25) A549 human lung cancer cells were cultured in DMEM cell culture fluid, so that the number of cells reached 110.sup.7; 110.sup.7 oncolytic adenoviruses were added to the culture fluid to infect human lung cancer cells; at 37 C. and 5% oxygen content, the infected tumor cells were cultured; at 48th hours after administration of oncolytic adenoviruses, when the tumor cells became smaller and dimmer, the supernatant of the human lung cancer cell culture fluid was centrifuged successively at the centrifugal force of 1000 g, 5000 g for each 10 minutes, the cells and debris were removed, and the supernatant after centrifugation was subjected to further centrifugation at the centrifugal force of 10000 g for 2 hours, precipitate was colletcted to obtain microparticles from cell vesicles derived from apoptotic A549 human lung cancer cells coating oncolytic adenoviruses.

(26) The microparticles prepared above were resuspended with 1 ml of 0.9% (g/ml) physiological saline. After fixed on glass, the microparticles were observed under transmission electron microscope, and the particle sizes of the microparticles were detected by Mastersizer 2000 particle size analyzer and the results were the same as Example 1. Fluorescent staining is used, the results also showed that the microparticles of this example coat oncolytic adenoviruses.

Example 4

(27) A549 human lung cancer cells were cultured in DMEM cell culture fluid, so that the number of cells reached 110.sup.7; 210.sup.8 oncolytic adenoviruses were added to the culture fluid to infect human lung cancer cells; at 37 C. and 5% oxygen content, the infected tumor cells were cultured; at 48th hour after administration of oncolytic adenoviruses, when the tumor cells became smaller and dimmer, the supernatant of the human lung cancer cell culture fluid was centrifuged successively at the centrifugal force of 1000 g and 5000 g for each 10 minutes, the cells and debris were removed, and the supernatant after centrifugation was subjected to further centrifugation at the centrifugal force of 10000 g for 2 hours, precipitate was collected to obtain microparticles generated from cell vesicles derived from apoptotic A549 human lung cancer cells coating oncolytic adenoviruses.

(28) The microparticles prepared above were resuspended with 1 ml of 0.9% (g/ml) physiological saline. After fixed on glass, the microparticles were observed under a transmission electron microscope, and the particle sizes of the microparticles were detected by Mastersizer 2000 particle size analyzer and the results were the same as Example 1. Fluorescent staining was used, the results also showed that the microparticles of this example coat oncolytic adenoviruses.

Example 5: Killing Effect of Oncolytic Adenovirus-Coated Microparticles on Different Tumor Cells

(29) 1. Experimental Steps

(30) The prepared oncolytic adenovirus-coated microparticles were respectively cultured with different types of human tumor cell lines which include A2780 human ovarian cancer cell line, MCF-7 human breast cancer cell line, A549 human lung cancer cell line, SNU1 human gastric cancer cell line, Caco2 human colon cancer cell line, HepG2 human hepatoma cell line, ASCP-1 human pancreatic cancer cell line, LINCap human prostate cancer cell line, as oncolytic adenovirus-coated microparticle treated groups. After 48 hours, observed the death condition of the cells. A group which treating the tumor cells with normal saline was control group, and a group which treating the tumor cells only with cell vesicles was used as cell vesicle-administered group.

(31) Preparation of oncolytic adenovirus-coated microparticles: microparticles of the above various tumor cells were obtained according to the method of Example 1.

(32) Microparticles of the above various tumor cells obtained according to the methods of Examples 3-4 also had the following similar tumor-killing effect.

(33) Preparation of cell vesicles: culturing tumor cells and irradiating with ultraviolet rays for 30 minutes. After 48 hours of UV irradiation, when tumor cells became obvious smaller and dimmer, centrifuging the supernatant of the mouse hepatoma cell culture fluid successively at the centrifugal force of 500 g, 1000 g and 5000 g for each 10 minutes, followed by centrifugation at the centrifugal force of 14000 g for 1 minute to remove cells and debris, and subjecting the supernatant after centrifugation to further centrifugation at the centrifugal force of 10000 g for 1 hour, collecting precipitate to obtain cell vesicles.

(34) 2. Experimental Results

(35) Tumor cells were treated as above, and after 48 hours, the cells in each group were observed under an inverted phase contrast microscope. The cells in control group and MP group (only administration of cell vesicles) showed an adherent growth state; while cells in OA-MP treated group (administration of oncolytic adenovirus-coated microparticles) were detached from adherence and showed a dead state (FIG. 3), indicating that the microparticles coating oncolytic adenoviruses have discernible cytotoxic effect on tumor cells.

Example 6: Microparticles Coating Oncolytic Adenoviruses have No Toxic Side Effects on Body

(36) 1. Experimental Steps

(37) Mice were tail vein injected with phosphate buffered saline (PBS), cellular vesicles (MP), oncolytic adenoviruses (OA) and oncolytic adenovirus-coated microparticles (OA-MP) one time a day respectively, total 5 times, the mice were killed on the 6th day, and then the content of glutamic-pyruvic transaminase (ALT) and creatinine (CRE) were detected in the serum of the mice. The microparticles and cell vesicles used in this example were the same as those in Example 2. Microparticles used in Examples 3-4 also had no toxic side effects on body.

(38) 2. Experimental Results

(39) As can be seen from FIG. 4a and FIG. 4b, compared with mouse injected with PBS or single cell vesicles in control group, there was no obvious change in the content of glutamic-pyruvic transaminase and creatinine for mouse injected with microparticles generated from cell vesicles coating oncolytic adenoviruses in treated group.

Example 7: Microparticles Coating Oncolytic Adenoviruses Inhibit Tumor Growth and Extend Survival Time of Tumor-Bearing Mice

(40) 1. Experimental Steps

(41) Nude mice were subcutaneously injected with 110.sup.6 A549 human lung cancer cells. After 5 days, cell vesicles (MP), oncolytic adenoviruses (OA) and oncolytic adenovirus-coated microparticles (OA-MP) were directly injected to a tumor inoculation site once for 3 days, total for 5 times. Observe the effect of subcutaneous tumor growth. The number of cell vesicles and microparticles for each injection were the same of 210.sup.6, the amount of adenoviruses was 210.sup.6.

(42) Nude mice were intraperitoneally injected with 510.sup.6 A549 human lung cancer cells. After 3 days, PBS solution, single oncolytic adenoviruses (OA), single cell vesicles (MP) and oncolytic adenovirus-coated microparticles (OA-MP) were directly intraperitoneally injected respectively once for 3 days, total for 5 times. Observe the mice survival time. The number of cell vesicles and microparticles for each injection were the same of 310.sup.6, the amount of adenovirus was 310.sup.6.

(43) Nude mice were intraperitoneally injected with 510.sup.6 A2780 human ovarian cancer cells. After 3 days, cell vesicles (MP), oncolytic adenoviruses (OA) and oncolytic adenovirus-coated microparticles (OA-MP) were intraperitoneally injected respectively once for 3 days, total for 7 times. Observe the intraperitoneal tumor growth. The number of cell vesicles and microparticles for each injection were the same of 310.sup.6, the amount of adenovirus was 310.sup.6.

(44) In this example, the method for preparing microparticles and cell vesicles was the same as Example 2. Using the preparation methods of Examples 3-4 would also obtain the following similar effects.

(45) 2. Experimental Results

(46) Nude mice were subcutaneously injected with A549 tumor cells, and tumor nodules began to grow on the 5th day. However, after the treatment of oncolytic adenoviruses-coated microparticles, the tumor growth was significantly inhibited, and the tumor volume was significantly smaller than that of the other control groups (FIG. 5a showed the size of tumor volumes on day 5 and day 21 and FIG. 5b showed tumor volumes were detected at different time points), i.e., compared to the group where only oncolytic adenoviruses were administrated and the group where single cell vesicles were administrated, the growth of subcutaneously A549 tumor of nude mice in group where oncolytic adenoviruses-coated microparticles were administrated, can be significantly inhibited.

(47) After nude mice were intraperitoneally injected with A549 tumor cells, the treatment of oncolytic adenovirus-coated microparticles significantly prolonged the lifetime of tumor-bearing mice. Compared to the group where single oncolytic adenoviruses were administrated, p<0.05 (FIG. 5c showed the survival rate of tumor-bearing mice after being treated with PBS, MP, OA, and OA-MP), that is, compared to the group where single oncolytic adenoviruses were administered and the group where single cell vesicleses were administered, the lifetime of tumor-bearing mice in the group where oncolytic adenovirus-coated microparticles were administrated can be significantly prolonged.

(48) After nude mice were intraperitoneally injected with A2780 tumor cells, then were treated with phosphate buffered saline (PBS), single cell vesicles (MP), single oncolytic adenoviruses (OA), and oncolytic adenovirus-coated microparticles (OA-MP) respectively, after 30 days, the tumor size and weight were detected and the results were shown in FIG. 5d, i.e. compared to the group where only oncolytic adenoviruses were administered and the group where single cell vesicles were administered, the treatment of the group where oncolytic adenovirus-coated microparticles were administrated can significantly inhibited the growth of ovarian tumor. Compared to the single oncolytic adenovirus group (see FIG. 5d), p<0.05.