CN108753738B - Oncolytic virus and application thereof - Google Patents
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Abstract
The invention discloses an oncolytic virus and application thereof, and relates to the technical field of biology. The preservation number of the oncolytic virus is CCTCC No. V201809. The oncolytic virus has an anti-tumor effect, and can be used for preparing an anti-tumor medicament with an excellent anti-tumor effect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an oncolytic virus and application thereof.
Background
Oncolytic viruses (Oncolytic viruses) are a class of viruses that preferentially infect and kill tumor cells. Initially, a portion of the tumor cells are specifically infected and destroyed by the oncolytic virus. Subsequently, the oncolytic virus replicates and multiplies in tumor cells, releasing new infectious viral particles to infect and destroy other tumor cells. Oncolytic viruses exert their oncolytic effect by directly lysing tumor cells or stimulating the host to produce an anti-tumor immune response. The potential of viruses as antitumor drugs was discovered in the early 20 th century, but extensive research and development was not formally conducted until the 60's of the 20 th century.
At present, many viruses including adenovirus, reovirus, measles virus, herpes simplex virus, newcastle disease virus and vaccinia virus are clinically detected as oncolytic agents, and only a few viruses in nature can naturally generate oncolytic (such as reovirus, seneca virus and newcastle disease virus). The first oncolytic virus approved by the national regulatory agency was the genetically modified ECHO-7 enterovirus, RIGVIR, approved in 2004 in latavia for the treatment of cutaneous melanoma. Later (2015 and 2016, respectively), gurugia (nationwide) and amanita also approved this regulation. In 2005, shanghai three-dimensional (Sunway) biotechnology limited of china corporation registered a genetically modified oncolytic adenovirus named H101 and obtained regulatory approval of the national food and drug administration for the treatment of head and neck tumors. In 2015, the USFDA and EMA of the european union approved T-VEC as the first oncolytic herpes virus (a modified herpes simplex virus) for the treatment of melanoma, which is also the most successful oncolytic virus at present.
Newcastle disease virus belongs to the avian paramyxoviridae family, is a lethal virus of birds, but causes mild influenza-like symptoms or conjunctivitis in human beings. Newcastle disease viruses can spread cytotoxic viruses to every cancer cell of the body by viral replication, but the production of an immune system of virus neutralizing antibodies may limit this possibility.
Disclosure of Invention
The first purpose of the invention is to provide an oncolytic virus which has oncolytic capacity and can be used for preparing antitumor drugs.
The second objective of the present invention is to provide a vector, which can load oncolytic virus, precisely target tumor cells, and has the advantages of promoting virus-specific immune response and enhancing anti-tumor effect.
It is a third object of the present invention to provide a method for producing an immunoeffector cell loaded with an oncolytic virus, which can produce an immunoeffector cell having an oncolytic effect.
The fourth purpose of the present invention is to provide an application of the above-mentioned vector or the above-mentioned oncolytic virus in the preparation of an anti-tumor drug, which can prepare an anti-tumor drug that can precisely target a tumor and has a superior anti-tumor effect.
The fifth object of the present invention is to provide an antitumor agent having an advantage of excellent antitumor effect.
The invention is realized by the following steps:
the invention provides an oncolytic virus with a preservation number of CCTCC NO V201809.
The present invention provides a vector loaded with the above-mentioned oncolytic virus.
The invention provides a preparation method of an immune effector cell loaded with oncolytic virus. The preparation method comprises the step of contacting an oncolytic virus solution with an immune effector cell solution, wherein the oncolytic virus in the oncolytic virus solution is the oncolytic virus.
The invention provides an application of the oncolytic virus or the vector in preparation of an anti-tumor drug.
The invention provides an anti-tumor drug which comprises the oncolytic virus or the vector.
The invention has the following beneficial effects:
the oncolytic virus provided by the embodiment of the invention has a preservation number of CCTCC number V201809. The oncolytic virus has oncolytic capacity, and can be used for preparing antitumor drugs with excellent antitumor effect.
The vector containing the oncolytic virus provided by the embodiment of the invention can transfer the oncolytic virus, accurately targets tumor cells, and has the advantages of promoting virus specific immune response and enhancing anti-tumor effect.
The preparation method of the immune effector cell loaded with the oncolytic virus provided by the embodiment of the invention comprises the step of easily contacting the oncolytic virus with the immune effector cell, and the method can be used for preparing the immune effector cell with good targeting and excellent anti-tumor effect.
The embodiment of the invention provides application of the oncolytic virus or the vector in preparation of an anti-tumor medicament. The application can prepare the antitumor drug with excellent antitumor effect.
The anti-tumor drug provided by the embodiment of the invention comprises the oncolytic virus or the vector, and has the advantages of accurate targeting of tumors and excellent anti-tumor effect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph showing the results of confocal focusing of oncolytic virus-loaded immune effector cells according to a second embodiment of the present invention;
FIG. 2 is a graph of cell survival after infection of lung cancer cells by NDV/FMW as provided in the third embodiment of the present invention;
FIG. 3 is a graph of cell survival following infection of thyroid cancer cells with NDV/FMW as provided by the third embodiment of the present invention;
FIG. 4 is a graph of cell survival after infection of human embryonic lung fibroblasts and CIK cells by NDV/FMW as provided in the third embodiment of the present invention;
FIG. 5 shows CD8 in CIK cells after loading with NDV according to a third embodiment of the present invention+T cell and CD3+CD56+A change result graph of the NKT cell;
FIG. 6 is a graph showing the results of the change in the volume of subcutaneous tumors in nude mice according to the fourth embodiment of the present invention;
FIG. 7 is a graph showing HE staining results of CIK, NDV/FMW and NDV/FMW + CIK provided by a fifth embodiment of the present invention;
FIG. 8 is a graph showing the results of the NDV replication ability in tumor cells and CIK cells according to the sixth embodiment of the present invention;
FIG. 9 is a graph showing the result of the NDV loading efficiency of CIK cells measured by flow cytometry according to the first comparative example of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products available commercially.
The oncolytic virus according to an embodiment of the present invention and the use thereof will be specifically described below.
In one aspect, embodiments of the present invention provide an oncolytic virus, specifically a newcastle disease virus NDV/FMW strain, which has been deposited at the chinese type culture collection of date 2/6 of 2018, address: wuhan university in Wuhan, China, the preservation number: CCTCC NO V201809. The oncolytic virus has oncolytic capacity and can be used for preparing antitumor drugs. The oncolytic capacity of the oncolytic virus can be reduced by injecting the oncolytic virus solution into tumor cell tissues, so that the oncolytic virus has the oncolytic capacity on the tumor tissues.
Oncolytic viruses are a class of tumor-killing viruses with replication ability that can exert anti-tumor effects through a variety of mechanisms. However, the immune surveillance of the body does not allow the oncolytic virus to reach the tumor site efficiently enough to treat the tumor. Accordingly, embodiments of the present invention provide a vector loaded with the above-described oncolytic virus. The cell vector acts as a container for the oncolytic virus, so that the oncolytic virus avoids immune surveillance, and the cell vector and the oncolytic virus are combined to have better anti-tumor efficacy.
The vector comprises one or more of immune effector cells, mesenchymal stem cells, endothelial cells and tumor cells. Preferably, the immune effector cells may include antigen-specific T cells, cytokine-induced killer cells, tumor-associated macrophages, peripheral blood lymphocytes, and dendritic cells.
Preferably, the immune effector cell is a CIK cell. The CIK cell, named Cytokine induced killer in English, is a novel immunocompetent cell, has strong proliferation capacity and strong cytotoxic effect, and has certain immunological characteristics. The CIK cell has strong recognition capability on the tumor cell, can accurately target the tumor cell, but does not hurt normal cells, and has wide tumor killing spectrum and good safety. The CIK cell loaded with the oncolytic virus combines the tumor targeting property of the CIK cell and the tumor killing power of the oncolytic virus, and achieves a better anti-tumor effect.
The vector loaded with the oncolytic virus can enhance the oncolytic capacity of the vector and improve the anti-tumor effect of the vector on the basis of targeting tumor capacity. The embodiment of the invention also provides a preparation method of the immune effector cells loaded with the oncolytic virus, which comprises the following steps:
the preparation method comprises the step of contacting an oncolytic virus solution with an immune effector cell solution, wherein the oncolytic virus in the oncolytic virus solution is the oncolytic virus NDV/FMW provided by the embodiment of the invention.
Further, the multiplicity of infection by oncolytic virus is 10-100 MOI, and the cell density of immune effector cell solution is 1 x 106～5ⅹ106cells/mL.
The multiplicity of infection of the oncolytic virus can be: 10. 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 MOI.
The cell density of the immune effector cell solution may be: 1 x 106cell/mL, 1.5 x 106cell/mL, 2 x 106cell/mL, 2.5 x 106cell/mL, 3 x 106cell/mL, 3.5 x 106cell/mL, 4 x 106cell/mL, 4.5 x 106cell/mL or 5 x 106cells/mL.
When the multiplicity of infection of the oncolytic virus is 10-100 MOI, the cell density of the immune effector cell solution is 1 x 106～5ⅹ106At cell/mL, the oncolytic virus is able to be carried in maximal amounts by CIK cells.
In addition, the invention also provides application of the oncolytic virus or the vector in preparation of antitumor drugs.
The embodiment of the invention also provides an anti-tumor medicament which comprises the oncolytic virus or the immune effector cell.
The features and properties of the present invention are described in further detail below with reference to examples.
First embodiment
This example provides a method for preparing an oncolytic virus-loaded immune effector cell (CIK cell).
Induction and proliferation of CIK cells
Experiment consumable
Experimental methods
First, the experiment was prepared and UV-sterilized for 30 min. And turning on an air exhaust and illumination switch of the clean bench, and wiping the bench surface by 75% alcohol.
Collecting blood and routine examining the leucocyte in the peripheral blood is not less than 3X 106mL, lymphocyte count ≥ 6X 10550mL of peripheral blood. And (3) taking a 50mL centrifuge tube, slowly injecting the collected peripheral blood into the centrifuge tube, carrying out whole blood centrifugation, and centrifuging for 10min at the temperature of 20 ℃ at 2000 r/min. After the centrifugation is finished, sucking the upper plasma into a 50mL centrifuge tube, inactivating at 56 ℃ for 30min for later use.
Adding physiological saline into the centrifuge tube with the blood cells to 50mL, and slowly sucking, blowing and mixing the mixture uniformly by a suction tube. Slowly adding the uniformly mixed blood into the upper layer of the liquid surface of the lymphocyte separation liquid to ensure that the layering is clear, and centrifuging at 20 ℃ for 20min at 800 g.
The single nuclear cell layer (milky white cell ring) in the two tube separation liquid is collected to a 50mL centrifugal tube, physiological saline is added to the centrifugal tube to reach 50mL, and the centrifugal tube is centrifuged (20 ℃, 2000r/min, 8 min). Then, the supernatant was discarded, shaken, added with physiological saline to 50mL, centrifuged (20 ℃ C., 2000r/min for 5min), and washed twice. Discarding the supernatant, shaking, adding a certain amount of cell culture solution into the centrifuge tube, uniformly blowing and sucking the cells by using a suction tube, transferring the cells into a cell culture bottle, and brushing the centrifuge tube with the culture solution once.
Culture bottleThe total amount of the liquid is about 50mL, 2.5mL of inactivated plasma and gamma-interferon are added, and the final concentration of the gamma-interferon is 1000IU/mL and the mixture is mixed evenly. Observing cell number and state under microscope, placing at saturated humidity, 37 deg.C, and 5% CO2In the incubator, the culture was performed for 24 hours.
The next day, the cells cultured for 24 hours were taken out from the incubator, and the color of the culture solution, the transparency of the culture solution, and the number and state of the cells were observed under a mirror. Adding a mixed factor (CD3 antibody final concentration: 100 ng/mL; IL-2 final concentration: 1000 IU/mL; IL-1 alpha final concentration: 1ng/mL), placing at saturated humidity, 37 deg.C, and 5% CO2And (5) continuing culturing in the incubator.
And on the fifth day, the color of the culture solution is observed by naked eyes, the number and the growth state of the cells are observed under a mirror, and the activity of the CIK cells is confirmed. The confirmed cells were transferred to two cell culture flasks, and the medium containing IL-21000U/mL was placed at a saturated humidity of 37 ℃ and 5.0% CO2And (5) continuously culturing in the incubator.
On the fifteenth day, CIK cells were obtained that could be used for NDV loading.
Identification of CIK cells
The CIK cells in the culture bottle are colorless and semitransparent cell suspension. The cell survival rate is not lower than 90% by adopting an automatic cell counter trypan blue staining method. Detection Using flow cytometry, CD3+CD56+And CD3-CD56+The sum of the amounts is 20% or more. By adopting the CCK-8 kit, when the effective-target ratio is 20:1, the killing rate of CIK cells on leukemia cell strains K562 is ensured to be more than 50%.
Culturing the CIK cells obtained by separation in vitro for 14 days, washing the CIK cells with PBS three times, and then resuspending the CIK cells with X-VIVO.15 serum-free culture medium to adjust the cell density to 1 multiplied by 106～1× 107The cell survival rate is not less than 90 percent per mL.
Adjusting the concentration of CIK cell solution to 5 x 106cell/mL, mixing the oncolytic virus solution containing the oncolytic virus (FMW) with CIK cell solution at 10MOI to obtain mixed solution, incubating the mixed solution at 37 deg.C for 4 hr, washing away unbound virus with PBS, and removing X-VIVO.15After the serum medium is resuspended, the immune effector cells loaded with oncolytic virus are obtained.
Second embodiment
It was verified that the immune effector cells prepared by the preparation method of the first example were loaded with oncolytic virus.
Experimental methods
The immune effector cell CIK cells loaded with oncolytic virus provided in the first embodiment are observed by a laser confocal microscope. Specifically, a six-well plate was loaded with cell slide, inoculated with CIK cell suspension (2 mL/well), placed in an incubator for pre-culture for 24 hours, and 10MOI oncolytic virus (NDV/FMW) infected CIK cells for 4 hours at 37 ℃.
Taking out the slide, and washing with PBS for 2 times, 5 minutes each time; fixing with 4% paraformaldehyde for 15min, washing with PBS for 2 times, 5min each time; permeabilization of 0.03% Triton 100 for 10min, washing 2 times with PBS, 5min each time; HN (NDV virus protein) antibody was incubated overnight at 4 ℃ and washed 2 times with PBS for 5 minutes each; incubating goat anti-rabbit red fluorescent secondary antibody for 30 minutes at room temperature, and washing with PBS for 2 times, 5 minutes each time; DAPI staining for 10min, PBS washing 2 times, 5min each; and (3) sealing the chip by using a sealing agent containing a quenching agent, and observing the distribution of the HN protein in a confocal manner.
Results of the experiment
The confocal results are shown in figure 1, and it can be seen from figure 1 that NDV virus (oncolytic virus) protein HN is distributed on the surface of cell membrane, and the results show that NDV is successfully loaded on the surface of CIK cell.
Third embodiment
And verifying the effect on CIK cells after loading the oncolytic virus.
Cell survival effects of NDV Loading on tumor cells and on CIK cells
Experimental methods
Cell viability was measured using the CCK8 Kit (Cell Counting Kit 8). Specifically, a cell suspension (100. mu.L/well) was seeded in a 96-well plate and placed in an incubator for pre-culture for 24 hours. (the cell suspensions herein are lung cancer cells (A549 and H460), thyroid adenocarcinoma cells (THJ-16T and THJ-29T), human embryonic lung fibroblast (MRC-5), CIK cells. Different MOIs (0.001, 0.01, 0.1, 1) NDV/FMW infected lung cancer cells (A549 and H460), thyroid cancer cells (THJ-16T and THJ-29T), human embryonic lung fibroblasts (MRC-5), CIK cells 24, 48 and 72 hours. 10uL of CCK8 solution was added to each well 1 hour in advance and incubation continued for 1 hour. Absorbance at 450nM was measured with a microplate reader. Among them, MOI (multiplicity of infection, and ratio of number of virus particles/number of cells). The experimental results are shown in the attached figures 2-4.
Results of the experiment
According to the attached figures 2-4, NDV can obviously inhibit the proliferation of various tumor cells, which shows that NDV has strong oncolytic capacity, and NDV has no inhibition effect on the growth of normal embryonic lung fibroblast (MRC-5) and CIK cells, which shows that NDV does not influence the proliferation of normal cells.
CD8 in CIK cells loaded with NDV+Effect of T and NKT cells
Experimental methods
A6 cm dish was inoculated with CIK cell suspension (4 mL/dish) and placed in an incubator for preculture for 24 hours. 10MOI NDV/FMW infected CIK cells for 4 hours at 37 ℃. CIK cells infected and not infected with NDV were collected separately and centrifuged (2000 rpm, five minutes) to remove the supernatant. CD8 antibody was added, or CD3 and CD56 antibodies were added. Flow sorting CD8+Or CD3+And CD56+The ratio of the CIK cells before and after loading NDV was calculated. The results are shown in FIG. 5.
Results of the experiment
As can be seen from FIG. 5, CD8 appears before and after loading of NDV+The number of T cells and NKT cells was unchanged, indicating that the NDV loading did not affect the activity of CIK cells.
Fourth embodiment
The tumor-dissolving ability of the NDV-loaded CIK cells prepared by the preparation method of the first example was verified.
Experimental methods
40 nude mice of 6 weeks old were taken and injected with H460 cells (1X 10)7Only). When the subcutaneous tumor of the nude mouse reaches 200cm2(about 14 days), 40 tumor-bearing nude mice were randomly divided into 4 groups of 10 mice each. Respectively is A: PBS blank control group; b: CIK treatment group; c: NDV/FMA treatment group of W; d: NDV/FMW transshipment CIK (NDV/FMW-CIK) treatment group. Intratumoral injection, 2 times weekly, for 5 weeks of treatment. The subcutaneous tumor volume was measured every 5 days for a total of 40 days and plotted as shown in figure 6.
Results of the experiment
As can be seen from FIG. 6, CIK (NDV/FMW-CIK) treatment with NDV/FMW showed the strongest tumor growth inhibition in the CIK treated group and the NDV/FMW treated group.
Fifth embodiment
This example provides immunohistochemical methods to examine the effect of NDV-loaded CIK cells prepared by the preparation method of the first example on mouse tumors.
Experimental methods
20 nude mice of 6 weeks old were taken and injected with H460 cells (1X 10)7Only). When the subcutaneous tumor of the nude mouse reaches 200cm220 tumor-bearing nude mice were randomly divided into 4 groups of 5 mice each. Respectively is A: PBS blank control group; b: CIK treatment group; c: NDV/FMW treatment group; d: NDV/FMW transshipment CIK (NDV/FMW-CIK) treatment group. Intratumoral injection, 2 times per week, 3 weeks of treatment. After 3 weeks, the tumor-bearing nude mice were anesthetized and euthanized, and tumor tissues were taken to prepare pathological sections. Adopting hematoxylin-eosin (HE) staining method, the HE staining procedure is as follows:
paraffin sections were placed in a 60 ℃ oven, deparaffinized 4 hours, washed 3 times with PBS, 5 minutes each. The slices were processed according to the following procedure: xylene (I)5min → xylene (II) 5min → 100% ethanol 2min → 95% ethanol 1min → 80% ethanol 1min → 75% ethanol 1min → distilled water washing for 5 min. Adding hematoxylin dropwise for staining for 5min, and washing with running water. Dropwise adding hydrochloric acid and ethanol for differentiation for 30s, soaking in distilled water for 15min, dropwise adding eosin solution for dyeing for 2min, dehydrating conventionally, and sealing: 95% ethanol (I) min → 95% ethanol (II) 1min → 100% ethanol (I)1min → 100% ethanol (II) 1min → xylenecarbonate (3: 1)1min → xylene (I)1min → xylene (II) 1min → neutral resin seal.
Immunohistochemical detection of CD3 Positive Rate (indicating CIK cells)
Immunohistochemistry step:
paraffin sections were placed in a 60 ℃ oven, deparaffinized 4 hours, washed 3 times with PBS, 5 minutes each. Heating a citrate buffer solution with the pH value of 6.0 to boiling, putting the dewaxed and hydrated slices into the boiled buffer solution, performing microwave treatment for 10 minutes, naturally cooling by flowing water, and washing by PBS for 3 times and 5 minutes each time. 3% H was added dropwise to the slices2O2Incubate for 10 minutes at room temperature, and wash 3 times with PBS for 5 minutes each. PBS was blotted dry, each drop of CD3 antibody (1: 200), incubated at room temperature for 3 hours, and washed 3 times with PBS, each for 5 minutes. And (3) dripping horseradish peroxidase-labeled goat anti-rabbit secondary antibody, incubating for 30 minutes at room temperature, and washing for 5 minutes each time for 3 times by using PBS. The PBS solution was blotted dry, and freshly prepared DAB solution (diaminobenzidine) was added dropwise and observed under a microscope for 5 minutes. Then hematoxylin counterstain, 0.1% HCl differentiation, tap water rinse, bluing, slicing through 95% ethanol (I) min → 95% ethanol (II) 1min → 100% ethanol (I)1min → 100% ethanol (II) 1min → xylylen carbonate (3: 1)1min → ditoluene (I)1min → xylene (II) 1min → neutral resin sealing piece.
Results of the experiment
The HE staining results are shown in FIG. 7, and it is understood from FIG. 7 that CIK, NDV/FMW and NDV/FMW + CIK show tumor necrosis. The staining of CD3 antibody (used as CIK cell indicator) shows that CD3 expression can be detected in mouse sections of CIK and NDV/FMW + CIK treatment groups, and the number of CD3 in NDV/FMW is greatly larger than that of CD3 in CIK, thus the fusion of NDV/FMW and CIK can effectively increase the immunogenicity and effectively enhance the anti-tumor capacity of cells.
Sixth embodiment
The replication capacity of NDV in tumor cells and CIK cells is verified.
Experimental methods
Inoculating cell suspension (2 mL/well) into 6-well plate, placing into incubator, and pre-culturing for 24 hr (the cell suspension here is 6000/well, lung cancer cells (A549 and H460), thyroid cancer cells (THJ-16T and THJ-29T), and CIK cells) 0.01MOI NDV/FMW infected lung cancer cells (A549 and H460), thyroid adenocarcinoma cells (THJ-16T and THJ-29T), human embryonic lung fibroblast (MRC-5), and CIK cells 24, 48 and 72 hours. Cells and cell supernatants were collected and frozen 3 times, and supernatants (test virus solutions) were collected by centrifugation. DF1 cell (host cells of NDV, used to measure viral titer) suspensions (100 uL/well) were inoculated into 96-well plates and placed in incubators for pre-incubation for 24 hours. Diluting the virus liquid to be tested by 10 times (10)-1-10-11) Adding 8 multiple wells of each grads virus into each row of 96-well plate, and culturing (5% CO)237 ℃); and observing the lesion after 5-7 days, calculating the virus titer, and accurately calculating the titer by using a Reed-Muench two-stage method, wherein the experimental result is shown in the attached figure 8.
Results of the experiment
Wherein, for CIK cells, when the dilution of the virus solution to be detected is 10-8When the product is diluted to 10%, 75% positive is appeared-920% positive appeared with a distance ratio (percentage of lesion rate above 50% -50%)/(percentage of lesion rate above 50% — percentage of lesion rate below 50%), i.e. a distance ratio (75% -50%)/(75% -25%) 0.5. For 100 μ L of dilution lgTCID50 ═ distance ratio × logarithm of dilution + logarithm of dilution above 50% disease rate, i.e. lgTCID50 ═ 0.5 × (-1) + (-8) — -7.5/100uL, TCID50 ≈ 108.5/100 μ L ≈ 3.16 × 10 μ L ≈ 109.5/mL9TCID50/mL was converted to PFU/mL (this is the unit of viral particle): viral titer 3.16 × 109TCID50/mL ×0.7＝2.2×109PFU/mL。
As can be seen from fig. 8, NDV replicates in lung cancer cells as well as thyroid cancer cells and is time dependent. NDV can replicate in CIK cells as well, but is less able to replicate than in tumor cells. It is demonstrated that NDV can replicate and survive in CIK, but does not kill CIK cells on a large scale.
First comparative example
The multiplicity of infection of NDV in the preparation provided in the first example was verified.
Experimental methods
CIK cells (with the activity reaching more than 90%) prepared by the preparation method provided by the first embodiment are collected, centrifuged at 2000r/min for 5min, and the cells are repeatedly washed by PBS for 2 times. Serum-free medium resuspension of CIK cellsAnd adjusting the cell density to 1 × 106、5×106And 1 × 107Three groups respectively. NDV with the multiplicity of infection of 1MOI, 10MOI and 100MOI is respectively incubated with three groups of CIK cells with different cell densities for 4 hours at 37 ℃, unbound virus is washed away by PBS, and the cells are resuspended by serum-free culture medium to obtain the CIK cells loaded with the NDV. anti-NDV HN protein antibody was incubated for 1 hour at room temperature. And incubating FITC goat anti-rabbit IgG secondary antibody for 30min, and detecting the efficiency of NDV loading CIK cells by a flow cytometer, wherein the detection result is shown in figure 9.
As can be seen from FIG. 9, 5X 106CIK cells +10MOI NDV (88.5%) and 5X 106Both CIK cells +100MOI NDV (89.6%) groups were close to 90%, but 5X 106CIK cell +10MOI NDV combination ratio of 5X 106CIK cells +100MOI NDV combinations consumed less NDV, therefore, 5X 10 was selected6The combination of CIK cells +10MOI NDV is the optimal solution.
In summary, the oncolytic virus provided by the embodiment of the invention has a preservation number of CCTCC No. v201809. The oncolytic virus has oncolytic capacity, and can be used for preparing antitumor drugs with antitumor effect.
The vector containing the oncolytic virus provided by the embodiment of the invention can transfer the oncolytic virus, accurately targets tumor cells, and has the advantages of promoting virus specific immune response and enhancing anti-tumor effect.
The preparation method of the immune effector cell loaded with the oncolytic virus provided by the embodiment of the invention comprises the step of easily contacting the oncolytic virus with the immune effector cell, and the method can be used for preparing the immune effector cell with good targeting and excellent anti-tumor effect.
The embodiment of the invention provides application of the oncolytic virus or the vector in preparation of an anti-tumor medicament. The application can prepare the antitumor drug with excellent antitumor effect.
The anti-tumor drug provided by the embodiment of the invention comprises the oncolytic virus or the vector, and has the advantages of accurate targeting of tumors and excellent anti-tumor effect.
In addition, the invention also provides application of the vector or the oncolytic virus in preparing an anti-tumor medicament and the anti-tumor medicament comprising the oncolytic virus or the vector, and the anti-tumor medicament has the advantage of excellent anti-tumor effect.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. An oncolytic virus, which has a preservation number of CCTCC NO V201809.
2. A vector loaded with the oncolytic virus of claim 1.
3. The vector of claim 2, wherein the vector is an immune effector cell.
4. The vector of claim 3, wherein the immune effector cell is a CIK cell.
5. A method of producing an immune effector cell loaded with an oncolytic virus, the method comprising contacting an oncolytic virus solution with an immune effector cell solution, wherein the oncolytic virus in the oncolytic virus solution is the oncolytic virus of claim 1.
6. The method according to claim 5, wherein the multiplicity of infection of the oncolytic virus is 10 to 100MOI, and the cell density of the immune effector cell solution is 1X 106~5×106cells/mL.
7. The method according to claim 5 or 6, wherein the immune effector cell is a CIK cell.
8. Use of an oncolytic virus according to claim 1 or a vector according to any one of claims 2 to 4 for the preparation of an anti-tumor medicament.
9. An antitumor agent comprising the oncolytic virus according to claim 1 or the vector according to any one of claims 2 to 4.
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| Publication number | Priority date | Publication date | Assignee | Title |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (4)
| Title |
|---|
| Recombinant oncolytic Newcastle disease virus displays antitumor activities in anaplastic thyroid cancer cells;Ke Jiang et al.;《BMC Cancer》;20180718;第18卷;第1-10页 * |
| Specific immunotherapy in renal cancer: a systematic review;Armin Hirbod-Mobarakeh et al.;《Ther Adv Urol》;20171231;第9卷(第2期);第45-58页 * |
| 孟松树等.新城疫病毒 FMW 株体外溶瘤作用及其机制分析.《微生物学报》.2009,第49卷(第4期),第512-517页. * |
| 新城疫病毒 FMW 株体外溶瘤作用及其机制分析;孟松树等;《微生物学报》;20090404;第49卷(第4期);第512页摘要、第516页右栏第1段 * |
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