CN115518082A

CN115518082A - Coxsackie virus B group 5 type with oncolytic effect and application thereof

Info

Publication number: CN115518082A
Application number: CN202211145120.1A
Authority: CN
Inventors: 崔博沛; 宋丽芳; 高秋爽; 王茜; 吴星; 高帆; 刘明琛; 毛群颖; 梁争论
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2022-09-20
Filing date: 2022-09-20
Publication date: 2022-12-27

Abstract

The invention provides an application of CV-B5Faulkner strain in preparing human lung cancer tumor medicine, in particular to an application in improving the percentage of specific T cells and NK cells in tumor tissues and inducing CD4 in tumors ⁺ T cell, CD8 ⁺ Infiltration of T cells, NK cells. The invention also provides application of the strain in preparation of a medicine for treating tumors by combining with a DNA-PK inhibitor or an ATM inhibitor. The invention confirms that CV-B5/Faulkner does not infect normal lung tissue cells and can obviously kill lung cancer cell lines. For H460 cells with poor in vivo oncolytic effect, a remarkable ER stress-dependent synergistic enhancement effect can be exerted by combining a DNA-PK inhibitor or an ATM inhibitor. The invention uses protein waterExperiments on the level of the virus, the level of cells and the level in vivo prove that the virus has good selectivity, targeting property and therapeutic effect.

Description

Coxsackie virus B group 5 type with oncolytic effect and application thereof

Technical Field

The invention relates to the technical field of biological medicines and targeted medicine technology, in particular to a coxsackie virus B group 5 type with an oncolytic effect, and also relates to application of the virus in preparing medicines.

Background

Oncolytic viruses are viruses that are either naturally or genetically modified that can selectively replicate in tumor cells using the virus to lyse the tumor cells without damaging normal tissues. At present, four oncolytic viruses are listed worldwide and used for treating malignant melanoma, head and neck cancer and the like.

Enteroviruses (EV) are the main pathogens causing diseases such as infant hand-foot-and-mouth disease, herpanginSup>A, demethylation, myocarditis, aseptic meningitis and the like, and can be divided into 12 groups such as EV-A-H, J and rhinoviruses A-C and the like according to the gene and antigenic characteristics. Receptors for EV include poliovirus receptor (PVR), intercellular adhesion molecule-1 (ICAM-1), coxsackievirus and Adenovirus Receptor (CAR), decay Accelerating Factor (DAF), scavenger receptor 2, heparan sulfate, and the like. Related studies have demonstrated that certain EV receptors are overexpressed on the surface of certain tumor cells, conferring natural enterovirus oncolytic and selective killing effects. Enteroviruses having an oncolytic effect which have been found so far include coxsackievirus a21 (CV-a 21), an ECHO virus type 7 (ECHO 7), poliovirus (PV), coxsackievirus type B3 (CV-B3), and the like, in which ECHO 7 (trade name:

) Has been approved for marketing in Lasevia et al for the treatment of early malignant melanoma, CV-A21 (trade name:

) And PV have both entered clinical trials. Receptors of different kinds of EV are different, for example PV can infect and kill malignant glioma cells by using high-expression PVR, CV-A21 infects cell lines of breast cancer, colorectal cancer, pancreatic cancer and the like by using high-expression ICAM-1 and DAF, but has no toxicity or only weak toxicity to corresponding normal tissues.

After infecting host cells with oncolytic viruses through specific receptors, a large number of pathogen-associated molecular patterns and damage-associated molecular patterns are released through replication, innate immunity and adaptive immune responses are activated, a large number of cytokines and chemokines are produced, and lymphocytes are recruited to change the immunosuppressive state in tumor tissues. Research on the oncolytic effect of the coxsackievirus and an antigen detection reagent (Cui Bopei, 2020) screens and evaluates the oncolytic coxsackievirus, and finds that the coxsackievirus B group 5 (CV-B5) has a treatment effect in a nude mouse tumor-bearing animal model. However, tumor cells have abnormal activation of numerous signaling pathways, so that the oncolytic effect of viruses is greatly weakened, and research on inhibitors of the specific pathways combined with viruses is lacking at present.

Disclosure of Invention

The invention aims to provide an oncolytic virus having therapeutic significance on lung cancer tumors and verify the effect of the oncolytic virus and an inhibitor of a specific pathway in combination.

Based on the above, the invention provides the application of CV-B5Faulkner strain in the preparation of drugs for treating human lung cancer tumors, wherein the tumors also comprise liver cancer cells, pancreatic cancer cells, cervical cancer cells and the like.

In particular, it can be used for raising percentage of specific T cell and NK cell in tumor tissue and inducing CD4 in tumor ⁺ T cell, CD8 ⁺ Infiltration of T cells, NK cells.

On the other hand, the invention also provides application of the Coxsackie virus B group 5 type Faulkner strain and the DNA-PK inhibitor in preparing a medicament for treating tumors.

According to a preferred embodiment, the DNA-PK inhibitor is NU7441.

Preferably, such a drug is applied to the oncolytic expression of a p 53-expressing tumor cell, such as a human large cell lung cancer cell (NCI-H460).

The invention also provides application of the combination of the Coxsackie virus B group 5 type Faulkner strain and the ATM inhibitor in preparing a medicament for treating tumors.

According to another preferred embodiment, the ATM inhibitor is KU60019.

Similarly, the drug was applied to the oncolytic of tumor cells expressing p53, such as human large cell lung cancer cells (NCI-H460).

According to the invention, the oncolytic effects of coxsackie viruses in group A and group B, such as CV-B3/Nancy, CV-B3/112, CV-B5/Faulkner, CV-B5/JS417, CV-A6/Gdula and the like, are preliminarily researched through a cytotoxic experiment, and the result shows that except CV-A6 which only shows a weak oncolytic effect, the other four viruses can obviously kill lung cancer cells, liver cancer cells, cervical cancer cells and the like, but only CV-B5/Faulkner and CV-B5/JS417 do not infect normal lung tissue cells at 1MOI, and lung cancer cells such as A549, NCI-H1299 and NCI-H460 can be obviously killed at 0.1MOI, so CV-B5/Faulkner is selected for carrying out oncolytic research.

To further verify the screening results, we used 100MOI CV-B5/Faulkner to infect 2BS, MRC-5, KMB-17 and other normal lung tissue cells for 24h, and the cell survival rate was not statistically significant compared with the control group, and the mortality rates of 100, 10, 1, 0.1, 0.01, 0.001MOI CV-B5/Faulkner after infecting three lung cancer cells for 24h were dose-dependent.

Establishment of a mouse tumor model is a prerequisite for evaluation of the therapeutic effect of oncolytic virosomes. The BALB/c nude mouse CDX lung cancer model established by the invention adopts a virus intratumoral injection mode to evaluate the in vivo oncolytic effect of CV-B5. Experiments confirmed that CV-B5 (5X 10) was administered continuously 5 times when tumor diameter reached 4-5mm ⁶ TCID ₅₀ Needle) can completely inhibit or even cure the NCI-H1299 tumor, can inhibit the growth of the A549 tumor but rebound when the administration is stopped, and has no obvious treatment effect on the NCI-H460 tumor. When the NCI-H1299 tumor diameter reaches 4-5mm (early stage), 1 needle of CV-B5 (5X 10) is injected into the tumor ⁶ TCID ₅₀ ) The growth of the tumor can be completely cured, and no rebound phenomenon is observed after 100 days. When in useWhen the diameter of NCI-H1299 tumor reaches 8-9mm (late stage), 3 needles (5X 10) are injected into the tumor ⁶ TCID ₅₀ Needle) can inhibit the growth of tumor, the tumor volume can continuously increase at a slower speed after drug withdrawal than that of a control group, and the survival time of a treatment group is obviously longer than that of the control group. The oncolytic virus injected into the tumor can not only treat focus of infection parts, but also play a role in treating metastatic focus or multiple focus in a remote targeted way. When the tumor diameter reaches 4-5mm, CV-B5 is given for 5 times on one side to cure or obviously inhibit the growth of bilateral tumors. After the mice are cured by virus injected into tumor, the main organs do not have obvious pathological changes, and the toxic and side effects of CV-B5 are proved to be weak. Since it is difficult to evaluate the infiltration of immune cells induced by oncolytic virus due to immunodeficiency in nude mice, human peripheral blood mononuclear cells were injected intraperitoneally to beta ₂ m KO B-NDG mice an immune reconstituted animal model was obtained and used to study the effect of CV-B5/Faulkner treatment on the NCI-H1299 CDX model, suggesting that 5 consecutive 5-needles 5X 10 ⁶ TCID ₅₀ The CV-B5/Faulkner can obviously inhibit the growth of NCI-H1299 tumor tissues, and can induce obvious infiltration of CD3, CD4, CD8, granzyme B positive T cells and tumor cell apoptosis. In addition, due to the low expression of CAR in mouse Lewis Lung Cancer (LLC) and mouse colon cancer cell (ct26. Wt) cells, we constructed LLC-CAR-LUC and ct26.Wt-CAR-LUC cells by stable lentiviral transfection of CAR receptor and firefly Luciferase (LUC) to study the oncolytic effect and induced lymphocyte infiltration of CV-B5/Faulkner in naive immunocompetent mice. Experimental results show that the CV-B5/Faulkner has obviously better oncolytic effect on LLC-CAR-LUC and CT26.WT-CAR-LUC than wild LLC and CT26.WT cells, and CD4 in LLC-CAR-LUC after treatment ⁺ T cell, CD8 ⁺ The proportion of T cells and NK cells is obviously higher than that of the control group.

Since both the CDX model and the true tumor tissue are widely separated, we also constructed a PDX model using patient-derived tissues to further study oncolytic effects. The experimental results show that 5 needles in series are 5X 10 ⁶ TCID ₅₀ The CV-B5/Faulkner can obviously inhibit tumor bodiesThe product increases.

For H460 cells with poor in vivo oncolytic effect, a remarkable ER stress (ERS) -dependent synergistic enhancement effect can be achieved by using a DNA-PK inhibitor NU7441 or an ATM inhibitor KU60019 in combination with CV-B5. According to the invention, the oncolytic virus CV-B5 capable of selectively killing lung cancer cells is discovered through screening and comparison of cell levels, and the virus is proved to have good selectivity, targeting property and treatment effect through protein level, cell level and in vivo level experiments.

Drawings

FIG. 1 shows the oncolytic effects of CV-B3/Nancy, CV-B3/112, CV-B5/Faulkner, CV-B5/JS417, CV-A6/Gdula;

FIG. 2 shows the killing effect of CV-B5/Faulkner strain on normal lung tissue cells;

FIG. 3 shows the killing effect of CV-B5/Faulkner strain on lung cancer cells;

FIG. 4 shows the oncolytic effect and survival time of CV-B5 in the early stage of the tumor in nude mice bearing lung cancer;

FIG. 5 shows the therapeutic effect of the NCI-H1299 tumor-bearing nude mice of the early group at different times;

FIG. 6 shows the therapeutic effect and survival time of the NCI-H1299 tumor-bearing nude mice in the middle and late stage groups;

FIG. 7 shows the effect of CV-B5 on the distal targeting of NCI-H1299 and A549 tumor-bearing nude mice;

FIG. 8 shows histopathological changes of the major organs of nude mice after intratumoral injection of CV-B5;

FIG. 9 is a graph of the effect of CV-B5/Faulkner on LLC or CT.26WT in vivo and in vitro oncolytic effects on CAR-stabilized and the percentage of specific T cells, NK cells in tissue;

FIG. 10 is a graph of the in vivo oncolytic effect of CV-B5/Faulkner on immuno-reconstituted B-NDG mice;

FIG. 11 is a graph showing the oncolytic effect of CV-B5/Faulkner on the PDX model;

FIG. 12 is a graph showing the oncolytic effect of CV-B5/Faulkner in combination with NU7441 on different types of tumor cells;

FIG. 13 shows the enhanced DNA fragmentation effect of CV-B5/Faulkner in combination with NU 7441;

FIG. 14 shows the enhanced DNA fragmentation effect of CV-B5/Faulkner in combination with KU 60019;

FIG. 15 shows CV-B5/Faulkner in combination with NU7441 enhances apoptosis and promotes viral replication;

FIG. 16 shows that CV-B5/Faulkner in combination with KU60019 enhanced apoptosis and facilitated viral replication;

FIG. 17 shows the changes in the p53, p21 pathway of the virus after CV-B5/Faulkner was used in combination with NU7441 or KU 60019;

FIG. 18 is CV-B5/Faulkner in combination with NU7441 to induce PERK activation;

FIG. 19 is a graph showing that CV-B5/Faulkner in combination with NU7441 induces activation downstream of PERK;

FIG. 20 shows the use of CHOP, elevated pJNK levels and cleavage by caspase 12 in combination with CV-B5/Faulkner and NU 7441;

FIG. 21 is a graph of the in vivo level of synergistic oncolytic effect of CV-B5/Faulkner in combination with NU7441 or KU60019.

Detailed Description

The following examples serve to explain the technical solution of the invention without limiting it.

In the present invention, "%" used for expressing concentrations or ratios is a volume percentage, unless otherwise specified.

In the present invention, reference is made to the following cells, which can be obtained commercially or from the assignee of the present invention, including: human non-small cell lung cancer cell (A549), human non-small cell lung cancer cell (NCI-H1299), human large cell lung cancer cell (NCI-H460), human hepatoma cell (HepG 2), human hepatoma cell (PLC/PRF/5), human hepatoma cell (Hep 3B), human cervical cancer cell (HeLa), human embryonic lung fibroblast (WI-38), human embryonic lung fibroblast (HFL-1), human embryonic lung fibroblast (MRC-5), rhesus monkey kidney cell (LLC-MK 2), african green monkey kidney cell (Vero), human Rhabdomyosarcoma cell (Rhabdomospora comal, RD), human pancreatic cancer cell (PANC-1), human pancreatic cancer cell (Bxpc-3), human pancreatic cancer cell (Capan-1) were preserved by the Chinese food and drug institute cell resource center; human normal hepatocytes L02, human normal hepatocytes MIHA were purchased from ATCC.

In the present invention, the following viruses are mentioned, which are available from the prior art or commercially available, and are subcultured using the corresponding cell line, repeatedly frozen and thawed 3 times after the cells are completely diseased, centrifuged for 15min at 4000rpm at 4 ℃, sterilized by filtration and frozen at-80 ℃. Cell culture median infectious dose (CCID) was measured according to the Reed-Muench method ₅₀ ). The basic information of the virus is shown in table 1 below:

TABLE 1 basic information on strains

In the invention, BALB/C nude mice, BALB/C mice and C57BL/6 mice of Specific-pathogen-free (SPF) grade are purchased from the resource center of laboratory animals of the Chinese food and drug testing institute; SPF grade NPI mice were purchased from Beijing Ai Dema Biotechnology, inc.; SPF grade B-NDG (. Beta.2mKO) mice were purchased from Peking Baiosai Tokyo pharmaceutical science and technology, inc.

In the present invention, the following reagents or kits are also contemplated:

TABLE 2 major reagents

In the present invention, the following media or reagent configurations are also contemplated:

(1) Immunoblot experiment blocking solution: weighing 5g of skimmed milk powder and dissolving in 100ml of TBST;

(2) Antibody working solution: diluting the mixture 1000 times by using a sealing solution of an immunoblotting experiment;

(3) Complete medium: 50ml FBS and 5ml streptomycin diabody were added to the corresponding 500ml MEM, DMEM or RPMI 1640 medium.

(4) Maintaining the culture medium: to a corresponding 500ml of MEM, DMEM or RPMI 1640 medium were added 10ml of FBS and 5ml of the penicillin-antagonist.

Example 1 pretreatment

1. Cell culture and passage

Cell recovery: the cells to be revived were removed from the liquid nitrogen tank and quickly placed in a 37 ℃ water bath and shaken rapidly until a completely thawed cell suspension was formed. In a biosafety cabinet, the cell suspension was diluted to 5ml with the corresponding complete medium (Table 3 below), centrifuged at 1000rpm for 5min, the supernatant was discarded, the cells were uniformly resuspended in the corresponding complete medium and transferred to T25 cell culture flasks at 37 ℃ and 5% CO ₂ And (5) culturing.

Cell passage: after the cell fusion degree reaches 90% (part of cells reaches 70-80%), removing the culture medium, rinsing with PBS for 2-3 times, adding appropriate amount of trypsin according to the digestion difficulty degree of local cells, and shaking uniformly. After the cells become wrinkled and begin to shed, add the corresponding complete medium to stop digestion, and blow and beat uniformly to disperse. The digested cells were collected, centrifuged at 1000rpm for 5min and the supernatant discarded. The cells were resuspended by adding an appropriate amount of complete medium and transferred to culture flasks at the passage ratio described in Table 3.

Freezing and storing cells: the frozen stock solution is prepared by 90 percent of FBS and 10 percent of DMSO by volume ratio. Cells were diluted to 5X 10 with frozen stock ⁶ -1×10 ⁷ The seeds/ml are divided into freezing tubes and placed in a freezing box at minus 80 ℃ overnight, and then transferred to a liquid nitrogen tank for storage the next day.

Cell counting: 20 μ l of the cell suspension to be counted is aspirated, added to the counting chamber and counted according to the instructions of the counter. The counted cells were diluted with the complete medium to the cell amount described in Table 3 and plated in a 6-well plate or a 96-well plate.

Table 3 shows the culture medium, passage ratio and plating amount of the cells

Example 2 selection of oncolytic Coxsackie viruses at the cellular level

Taking lung cancer cell lines, normal lung tissue cell lines, liver cancer cell lines, normal liver cell lines and cervical cancer cell lines to carry out screening experiments, wherein the screening experiments comprise 12 cells such as A549, NCI-H1299, NCI-H460, WI-38, HFL1, MRC-5, hep3B, hepG, PLC/PRF/5, L02, hela, MIHA and the like, and screening CV-B3/Nancy, CV-B3/112, CV-B5/Faulkner, CV-B5/JS417 and CV-A6/Gdula strains by applying a CCK experiment: the cells were plated in 96-well plates at the corresponding cell amounts shown in Table 3, and the viruses were fused with the cells to obtain the cell viability.

Specifically, the virus was serially diluted with maintenance medium of the corresponding cells (MOI =0.01, 1). After the cell fusion degree reached 70-80%, the medium was discarded, washed 3 times with PBS, and the PBS was discarded. The serially diluted virus was then added to a 96-well plate, triplicate wells were made for each dilution, triplicate Kong Yinxing controls (wells with cell growth, plus maintenance medium) and triplicate well blanks (wells without cell growth, plus maintenance medium) were set up and placed at 37 ℃ for 5% CO ₂ An incubator. After 24/48h, CCK8 reagent was added, 10. Mu.l per well, as OD in negative control well ₄₅₀ When the value reaches about 1.0, the OD is read ₄₅₀ The value is obtained.

OD of experimental well, negative control well and blank control well ₄₅₀ Values were averaged and substituted into the following formula to calculate the cell viability for each well:

the results of inhibition of 12 cells by 5 strains are shown in FIG. 1. The experimental results show that: except that CV-A6 only shows weak cytotoxicity, the other 4 strains of viruses can kill liver cancer cell lines to different degrees, but two CV-B3 strains can kill normal liver tissue cell lines strongly, and two CV-B5 strains have weak killing effect on normal liver cell lines, so that the hepatoma cell sensitive oncolytic virus cannot be used; both CV-B3 and CV-B5 killed the lung cancer cell lines strongly, but CV-B3 infected and lysed lung normal cell lines, whereas CV-B5 failed to kill normal lung tissue cells at MOI = 1. As the oncolytic effects of the two CV-B5 strains have no obvious difference, CV-B5/Faulkner is selected as the optimal oncolytic virus strain for subsequent experiments.

EXAMPLE 3 examination of the cytotoxicity of CV-B5 on a cell line of normal lung tissue

In the same experimental method as in example 2, with CV-B5/Faulkner and normal lung tissue cell line human embryonic lung diploid cells (WI-38), human embryonic lung diploid cells (HFL) and human embryonic lung fibroblast cells (MRC-5) as the subjects, the CCK8 experiment was performed to examine the cytotoxicity of CV-B5 on the normal lung tissue cell line by increasing the use concentration of CV-B5/Faulkner to 100MOI, and the results are shown in FIG. 2.

The results show that CV-B5/Faulkner at 100MOI still can not kill normal lung tissue cells, indicating that CV-B5/Faulkner has no cytotoxicity to normal lung tissue cell line.

Example 4 examination of the relationship between CV-B5 dose and oncolytic Effect of Lung cancer

In the same experimental method as in example 2, the dependence of CV-B5 dose on the oncolytic effect of lung cancer was examined by performing a CCK8 experiment using CV-B5/Faulkner as a target with respect to human non-small cell lung cancer cell (A549), human non-small cell lung cancer cell (NCI-H1299) and human large cell lung cancer cell (NCI-H460) and the concentration gradient of CV-B5/Faulkner as MOI =0.0001, 0.001, 0.01, 1, 10, 100.

The results are shown in FIG. 3, which shows that 10, 1, 0.1, 0.01, 0.001, 0.0001MOI CV-B5/Faulkner virus infected A549, NCI-H1299, NCI-H460 cells for 48H, and their cytolysis was found to be dose-dependent.

Example 5 evaluation of Effect of CV-B5/Faulkner application to Lung cancer-bearing nude mice

1. Establishment of tumor-bearing mouse model

(1) Human non-small cell lung cancer cells (A549), human non-small cell lung cancer cells (NCI-H1299) and human large cell lung cancer cells (NCI-H460) were mass-cultured in T225 cell culture flasks, respectively.

(2) Selecting 4-week-old female BALB/c nude mice, and implanting 5 × 10 mice subcutaneously in axillary region ⁶ And (4) cells.

(3) The diameter of the tumor can reach 4-5mm after 7-10 days; the diameter of the tumor can reach 15mm after the tumor is formed at 10-15d, and the nude mice are considered to reach the survival end point in ethics.

2. Oncolytic effect at early stage of tumor

The growth of tumors in mice has a growth pattern similar to that of humans, and initially undergoes a slow tumor volume increase (early stage), then enters a rapid tumor growth phase (middle stage), and to some extent enters a plateau stage (late stage). The evaluation of the oncolytic effect in a slow-growing period is an important index for evaluating the oncolytic effect in a virus body.

A549, NCI-H1299 and NCI-H460 tumor-bearing mice are obtained respectively. When the tumor diameter reaches 4-5mm, 5X 10 is injected into the tumor ⁶ TCID ₅₀ CV-B5, a control group is set up at the same time, each group is not less than 5, injection is carried out once every other day for five times, and tumor volume is measured and calculated continuously, and the ethical survival endpoint is taken as that the tumor diameter reaches 15 mm.

The major diameter and the minor diameter of the tumor were measured with a vernier caliper every other day from the first injection, and the tumor volume was calculated as follows:

as a result, as shown in FIG. 4, NCI-H1299 tumor-bearing mice were injected with 5CV-B5/Faulkner injections (5X 10 injections each) ⁶ TCID ₅₀ ) The tumor is completely cured after the treatment, and the tumor is continuously observed for 60 days without relapse; after 5CV-B5 injections, the tumor growth of the A549 tumor-bearing mice is obviously inhibited, but rebound occurs after the injection is stopped (namely after 10 days), but the survival period of the treatment group is prolonged by 5 to 10 days compared with that of the control group; the tumor growth rate and survival time of the NCI-H460 tumor-bearing mice after 5CV-B5 injections are not significantly different from those of the control group. Shows that CV-B5 has no obvious treatment effect on the mice with NCI-H460 tumor, the A549 tumor-bearing mice have limited treatment effect, and the mouse has obvious effect on the mice with NCI-H1299 tumor.

3. Tumor dissolving effect of different times of injection in early and middle and late stages of tumors

NCI-H1299 tumor-bearing mice were used as subjects.

Although the tumor growth is generally inhibited by continuously and repeatedly administering the oncolytic virus for the treatment at present, the pain of a patient can be greatly relieved by reducing the dosage of the oncolytic virus under the condition of not influencing the effect. Therefore, the tumor dissolving effect of NCI-H1299 tumor-bearing mice at different growth stages and different times is further examined.

NCI-H1299 tumor-bearing mice were obtained. Then, the early and middle late groups were set: when the tumor diameter of the early group reaches 4-5mm, dividing into 5, 4, 3, 2, 1 needle groups with no less than 5 per group, setting control group, and injecting 5 × 10 intratumorally ⁶ TCID ₅₀ Virus, once every other day; dividing into 5, 4, and 3 needle groups after the tumor diameter reaches 8-9mm, and setting control group with no less than 5 per group, injecting 5 × 10 intratumorally ⁶ TCID ₅₀ Virus was injected once every other day.

The results in the early group are shown in fig. 5, the tumor growth in all five groups is completely inhibited, the tumor in some mice is completely disappeared, and no rebound tendency is observed for 100 days.

The results for the middle and late stage groups are shown in fig. 6, and tumor growth was completely inhibited in the three groups. In addition, tumors in the middle and late stage groups rebound to different degrees within a period of time after treatment, and the survival curves are counted from the first treatment to the 60 th day of the tumors and are prepared by taking 15mm as an ethical survival endpoint, as shown in fig. 6, the survival times of mice in the 5, 4 and 3 needle treatment groups have no statistical difference, but are all obviously longer than those of the control group.

4. Evaluation of Effect of remote Targeted application of CV-B5

A549 and NCI-H1299 cell tumor-bearing mice are used as research objects.

On the basis of the fact that CV-B5 has the function of dissolving the tumor at the injection site, whether the oncolytic virus has the function of inhibiting the growth of the tumor at the non-injection site or not is examined, and therefore the feasibility of applying the oncolytic virus to metastatic or multiple cancers is examined.

The tumor-bearing mice NCI-H1299 and A549 with bilateral growing tumors were constructed in the same manner as described above, and female BALB/c nude mice of 4 weeks old were selected and implanted with 5X 10 tumors subcutaneously in bilateral underarm sites ⁶ And (4) one cell. When the tumor diameter reaches 4-5mm, 5X 10 is injected into the tumor on one side ⁶ TCID50Virus, control groups were established simultaneously, no less than 5 per group, and injections were administered five times every other day. The major and minor diameters of bilateral tumors were measured continuously.

The volume change curves of the bilateral tumors of the NCI-H1299, A549-treated group and the control group are shown in FIG. 7, which indicates that five consecutive single intratumoral injections of CV-B5 completely cured the bilateral tumors of the mice bearing the early NCI-H1299 tumor; the growth of bilateral tumors of A549 tumor-bearing mice can be obviously inhibited, but the tumor rebounds after the drug is stopped. The experimental result shows that CV-B5 can obviously inhibit the in vivo proliferation of lung cancer cells, but because different types of lung cancer cells have different reactivities, other medicines are required to be combined when the medicine is used for treating some types of lung cancer.

Example 6 histopathological changes in major mouse organs following intratumoral injection of CV-B5 in nude mouse CDX model

The lung, liver, brain, spleen, kidney, heart and acetyl pancreas tissues of nude mice injected with five needles in tumor were dissected, placed in neutral formalin solution, fixed to prepare tissue sections, and subjected to HE staining. As shown in FIG. 8, except for the slight histopathology at the site indicated by the arrow, significant histopathological changes were observed in all tissues, indicating that CV-B5/Faulkner is safe.

Example 7 the anti-tumor effect of CV-B5 is dependent on the expression of cell surface CAR and can induce significant immune cell infiltration

Tumor animal models established using nude mice are difficult to assess virus-induced tumor-localized immune cell infiltration, so animal models were constructed with LLC and ct.26wt cells at C57 or BALB/C, however, since both cells were low expressing CAR, we constructed LLC-CAR and CT26WT-CAR cell lines stably transfected with CAR using lentiviral vectors and confirmed stable expression of CAR using immunoblot experiments. The immunoblot assay procedure was as follows:

(1) Mu.l of PMSF was added to 100. Mu.l of RIPA to obtain a protein lysate. Adding 50 μ l protein lysate into the cell precipitate, and performing ice lysis for 20min while flicking for 2-3 times.

(2) Centrifuging at 12000rpm for 15min at 4 ℃, collecting supernatant, and determining the protein content by using a BCA method.

(3) Mixing the protein with 6 × SDS-PAGE loading buffer uniformly, and heating in boiling water bath for 15min.

(4) And (3) installing an electrophoresis tank, loading the denatured protein into the holes of the prefabricated gel, wherein the loading amount is 30 mu g/hole, and the loading amount of the protein marker is 10 mu l.

(5) Electrophoresis is carried out at constant voltage of 120V until bromocresol blue runs to the bottom of the gel, and the electrophoresis is stopped for about 50min.

(6) And (3) soaking the nitrocellulose membrane in the electrotransformation liquid for 1min, and sequentially placing the filter paper, the membrane, the gel and the filter paper on a protein molecule transfer instrument for electrotransformation for 15V 15min.

(7) And after the membrane is transferred, soaking the membrane in a sealing solution for immunoblotting experiments, and oscillating for 2 hours at room temperature.

(8) And diluting the antibody with a blocking solution according to the use instruction to obtain a primary antibody working solution.

(9) The membrane area to be detected was cut and soaked in the working solution and shaken overnight at 4 ℃.

(10) The antibody was discarded and the membrane was washed 3 times 10 min/time with TBST shaking.

(11) TBST was discarded, and HRP-labeled goat anti-mouse/goat anti-rabbit IgG was diluted 1000-fold to obtain a secondary antibody working solution, which was reacted at room temperature for 1 hour.

(12) Step 10 is repeated.

(13) And (4) preparing ECL developing solution freshly, developing at room temperature, and reading the detection result by using a multifunctional imager.

The oncolytic effect of CV-B5 was evaluated using an animal model, and the results are shown in fig. 9: CV-B5 had an in vivo oncolytic effect only on stably transfected LLC-CAR and CT26WT-CAR cells. Simultaneous study of CD3 in LLC-CAR tumor tissue using flow cytometry ⁺ CD4 ⁺ T cell, CD3 ⁺ CD8 ⁺ T cell, CD3 ⁺ NK1.1 ⁺ Percentage of NK cells, flow cytometry experimental procedure:

(1) Dissecting cancer tissue and digesting with collagenase 30min, centrifuging at 1000rpm for 10min, discarding supernatant, washing the pellet 3 times with PBS (containing 1% FBS), and separating the pellet from mononuclear cells with mouse lymphocyte separation solution. After 3 washes with PBS and counting, the cells were placed in ep tubes, with no less than one hundred thousand cells per tube.

(2) Taking CD3, CD4, CD8 and NK1.1 antibodies, and carrying out the following steps of 1:1000 concentration ratio was added to the above cell suspension, incubated 30min at room temperature in the dark, centrifuged at 1000rpm for 5min, washed 3 times with PBS (containing 1% FBS), and finally resuspended with PBS (containing 1% FBS). At the same time, a blank cell control tube and a monocell tube with not less than fifty thousand cells per tube are prepared.

(3) Blank tube cells were obtained with CELLQuest (BD, usa) software and the fluorescence voltage was adjusted; then adjusting fluorescence compensation by detecting the cells of the single anode tube one by one;

(4) 30000 cells from the sample tube were obtained and analyzed for the percentage of each type of cell.

As shown in FIG. 9, the intratumoral injection of CV-B5 significantly increased the percentage of specific T cells (CD 4, CD 8) and NK cells in tumor tissues.

EXAMPLE 8CV-B5 treatment of tumor-bearing immune reconstituted B-NDG mice induced intratumoral CD4 ⁺ T cell, CD8 ⁺ Significant infiltration of T cells, NK cells

In addition to the in vivo oncolytic effect of CV-B5 using BALB/C nude mice, C57 and BALB/C mice, we also used immuno-reconstituted B-NDG mice to evaluate the oncolytic effect and the infiltration of immune cells. Experiments were performed using beta 2 microglobulin knockout B-NDG mice, since wild type B-NDG mice develop graft-versus-host immune rejection after PBMC injection, leading to a shortened window period for the experiments. The specific method comprises the following steps: intraperitoneal injection of four-week-old B-NDG mice by 10 ⁷ PBMC in humans, one week later, 5X 10 axillary implant ⁶ NCI-H1299-Luc cells, when the tumor volume reaches 4-6mm, the intratumoral injection of virus 5X 10 is started ⁶ TCID ₅₀ The day is the next day (D2) and five virus treatments are given in succession at D4, 6, 8, 10; and fluorescence intensity was measured by intraperitoneal injection of firefly luciferase substrate at D3, 5, 7, 9, 11. At D12, the tumors were dissected, completely soaked in neutral formalin solution, tissue sections were prepared, and immunohistochemical staining was performed, including Cleaved caspase 3 (cleared-caspase 3), ki67, CD3, CD4, CD8, granzyme B (Granzyme B), and the like. The results are shown in FIG. 10, positive reaction of Ki67 in tumor tissue after CV-B5 treatmentThe degree is lower, namely the positive yellow staining degree is lighter; the positive reaction of the cut caspase 3 is more, and the result shows that the yellow stain of the whole specific cells is heavier. In addition, only significant CD3+, CD4+, CD8+, granzyme B positive cells were observed in the tumor tissue after CV-B5 treatment as indicated by the arrows. Thus, the increase in tumor volume was significantly inhibited in the treated group compared to the control group, mainly by induction of apoptosis and significant CD4 ⁺ 、CD8 ⁺ Infiltration of T cells.

EXAMPLE 9 therapeutic Effect of patient derived tissue model (PDX)

The PDX (Patient-derived xenotransplantation) model refers to a human tumor Xenograft model and is widely applied to current work. The PDX model is an animal model constructed by transplanting tumor tissues or cells of a patient into a mouse body and growing a human tumor in the mouse body. This model is currently an important tumor model because it can adequately reflect aspects of the original tumor. In the experiment, a PDX lung cancer animal model is constructed by using NPI mice, and when the tumor volume reaches 4-5mm, the intratumoral injection of CV-B5/Faulkner is started, wherein the injection is performed at a rate of 5 multiplied by 10 each time ⁶ TCID ₅₀ Once daily, five times total. And the change of the tumor volume is measured, the result is shown in figure 11, the tumors in the treatment group are obviously inhibited, and CV-B5/Faulkner has obvious inhibiting effect on the human tumors.

Example 10 the antitumor Effect of CV-B5 on insensitive cells synergistically enhanced by the combination of DNA-PK inhibitor NU7441 at cellular level

Based on the results of the foregoing example 4, CV-B5 alone had a weaker oncolytic effect on NCI-H460 cells than A549 and NCI-NCI-H1299 cells, and the in vivo experimental results of example 5 showed that five consecutive needle intratumoral injections had no significant therapeutic effect on NCI-H460. Therefore, by combining the inhibitors of DNA damage repair NU7441 or KU60019, it was investigated whether there was a synergistic effect.

Wherein, firstly, the dosage required by the virus used alone is determined so as to ensure that the killing rate to cells is lower without influencing the effect when the drugs are used together. As can be seen from the results of example 4, the survival rate of NCI-H460 cells treated by CV-B5/Faulkner at 0.01MOI was about 80%, and thus the dose was selected for the combination experiment. NU7441 was diluted to 10. Mu.M, 5. Mu.M, 2.5. Mu.M, 1.25. Mu.M, 0.625. Mu.M, 0.3125. Mu.M, NCI-H460 cells were infected at each dilution with 0.01MOI CV-B5/Faulkner, with different dilutions of the inhibitor (group 1) or a mixture of different dilutions of the inhibitor and 0.01MOICV-B5/Faulkner (group 2), and cell viability at different dilutions was obtained 24H later using the CCK8 experiment.

The cell viability of the inhibitor-treated group (group 1) and the combined group (group 2) at different dilutions were fitted to curves, and the area under the curve (AUC) was calculated. And recalculating the DAUC:

DAUC = [ AUC (group 2) -AUC (group 1) ]/AUC (group 1).

The DAUC in the NU7441 group was calculated to be 0.818, and the combination of the two drugs was considered to exert a synergistic effect.

In addition, the invention also compares the combined synergistic effect of 1 mu M NU7441 and different MOI CV-B5 on various cells, and the experimental result is shown in FIG. 12, NU7441 can enhance the oncolytic effect on p53 expressing cells (PANC-1, NCI-H460, capan-1, bxpc-3) but not on p53 deletion cells (NCI-H1299 and Hep 3B).

Example 11 cellular level in combination with NU7441 or KU60019 can significantly promote CV-B5/Faulkner-induced apoptosis

Reactive oxygen species induced by CV-B5/Faulkner replication in cells can promote double-stranded DNA fragmentation of host cell genome, and the process is marked by the increase of pH2AX protein level and promotes the phosphorylation of DNA-PK and/or ATM, thereby promoting the recombination repair of DNA at non-homologous ends or homologous recombination repair to avoid the death of host cells. NU7441 and KU60019 act on DNA-PK or ATM, 0.01MOI CV-B5/Faulkner and 1 μ M NU7441 or 1 μ M KU60019 are combined to act on NCI-H460 cells, and the phosphorylation levels of DNA-PK, ATM and H2AX in the cells and the protein expression level of the virus are detected by an immunoblotting experiment after 24H (the specific method is the same as the immunoblotting experiment step of example 7).

The results showed that intracellular phosphorylation of DNA-PK and ATM induced by viral replication could be inhibited by NU7441 or KU60019 and promoted an increase in pH2AX levels (fig. 13, 14). In addition, the combination of the two drugs can obviously promote the replication process of viruses in cells, which is shown in that the virus structural protein VP1 is increased, and the apoptosis effect caused by the viruses is promoted (figures 15 and 16).

Example 12 cellular levels in combination with NU7441 or KU60019 inhibit activation of the p53 pathway

The P53 gene is closely related to human tumor suppression. Activation of DNA-PK or ATM induces activation of the downstream p53 pathway, which is manifested by increased phosphorylation of p53 (ser 15), and accumulation of downstream p21 protein to promote cell cycle arrest and survival processes, which appear to be detrimental to CV-B5 function as an oncolytic agent.

The present invention confirmed the activation of downstream p53 pathway by immunoblotting (the same method as the immunoblotting procedure in example 7) of 1 μ M NU7441 or 1 μ M ATM with 0.01MOI CV-B5, and showed that the combined use of CV-B5/Faulkner and NU7441 or KU60019 could inhibit the activation of p53 and p21 by viruses, thereby promoting cell death (FIG. 17).

In the case of p 53-deficient cells, such as NCI-H1299 and Hep3B, the combination of NU7441 does not promote the activation of the death pathway, since CV-B5 does not induce the activation of the p53 pathway.

Example 13 cellular levels in combination with NU7441 promote activation of the PERK pathway and promote cell death

The virus can induce the unfolded protein reaction of endoplasmic reticulum by large-scale replication in cells, and specifically comprises three main channels, namely IRE1, PERK and ATF6 channels, wherein the three channels can promote the cells to generate ERS after inducing the activation of phosphorylated IRE1 (pIRE 1), phosphorylated PERK (pPERK) and ATF6 (P). If ERS of the cell is weak, the cell can relieve itself by inducing RIDD or peIF2 alpha; if a stronger ERS occurs, cell progression death can be promoted by induction of CHOP, caspase 12, pJNK, etc.

The invention detects the expression quantity of IRE1, pIRE1, PERK, pPERK and ATF6 (P) after the treatment of 0.01MOI CV-B5 and 1 mu M NU7441 for 24h through an immunoblotting experiment so as to reflect the activation condition of ERS.

The combined use of NU7441 and CV-B5/Faulkner in the present invention can induce activation of PERK pathway (phosphorylated p-PERK) instead of IRE1 and ATF6 pathways and promote activation of peRK downstream peIF2 alpha and ATF4, ultimately promoting elevation of CHOP, pJNK levels and cleavage of caspase 12 (caspase 12) (FIGS. 18, 19, 20).

Example 14 in vivo level experiments NU7441 or KU60019 show synergistic oncolytic enhancing action against CV-B5/Faulkner

Using the nude mouse animal model of example 5, intratumoral injection of drugs and viruses was started after tumor volume reached 4-5mm, as follows: NU7441 or KU60019 was administered at a dose of 10mg/kg/day by intraperitoneal injection at a dose of 5X 10 ⁶ TCID ₅₀ CV-B5/Faulkner, intratumorally injected once daily five times in a row, with inhibitor or virus alone as a control, by continuously measuring tumor volume, plotting the change of tumor volume over time, and comparing the effects of the combined in vivo treatments.

The results are shown in fig. 21, and the combination of NU7441 and KU60019 showed significant therapeutic effects.

Claims

1. Application of Coxsackie virus B group 5 in preparing medicine for treating lung cancer tumor is provided.

2. Use according to claim 1, characterized in that said medicament increases the percentage of specific T cells, NK cells within the tumor tissue.

3. Use according to claim 1, characterized in that the drug induces intratumoral CD4 ⁺ T cell, CD8 ⁺ Infiltration of T cells and NK cells.

4. The application of the combination of the Coxsackner virus B group 5 type Faulkner strain and a DNA-PK inhibitor in preparing a medicament for treating tumors.

5. The use according to claim 4, characterized in that the DNA-PK inhibitor is NU7441.

6. The use according to claim 4, characterized in that the medicament is applied for the oncolytic treatment of tumor cells expressing p 53.

7. The application of the Coxsackner virus B group 5 type Faulkner strain and the ATM inhibitor in preparing the medicine for treating the tumor.

8. Use according to claim 7, characterized in that the ATM inhibitor is KU60019.

9. The use according to claim 7, characterized in that the medicament is applied for the oncolytic lysis of tumor cells expressing p 53.