An anti-tumor virus

Abstract

The present invention discloses a virus for inhibiting Wnt signal and a method for inhibiting Wnt signal using the virus, and also discloses a method for treating tumors using the virus.

Description

Technical Field

[0001] The present invention relates to a virus for inhibiting Wnt signal and a method for inhibiting Wnt signal using the virus, and also relates to a method for treating tumors using the virus. Background Art

[0002] Hand, foot and mouth disease is an infectious disease that is common in children under 5 years old and is rarely seen in adults. The infection routes include the digestive tract, respiratory tract and contact transmission. The incubation period of hand, foot and mouth disease is 2-10 days, with an average of 3-5 days. Symptoms include mouth pain, anorexia, low fever, small blisters or ulcers on the hands, feet and mouth. Most children recover on their own within a week, while a few develop complications such as myocarditis, pulmonary edema, and aseptic meningoencephalitis. Some severe cases develop rapidly and lead to death. There is no effective treatment yet, and symptomatic treatment is the main treatment.

[0003] A variety of enteroviruses can cause hand, foot and mouth disease. The most common pathogens are Coxsackievirus A16 (CV-A16, also known as CVA16) and enterovirus 71 (EV-A71, also known as EV-71 or EV71), both of which belong to the genus Enterovirus in the family Picornaviridae.

[0004] Coxsackieviruses are divided into two types: A (23 types) and B (6 types). Although CV-A16 is an important pathogen of hand, foot and mouth disease, 90% of coxsackievirus infections do not cause symptoms or only fever.

[0005] To date, people's understanding of these enteroviruses is still limited, so it is necessary to further study the pathogenicity spectrum and pathogenesis of enteroviruses. Summary of the invention

[0006] In the process of studying the pathogenic spectrum of enteroviruses using Coxsackievirus A10 (CV-A10, also known as CVA10) as a model, the inventor unexpectedly discovered that enteroviruses such as CV-A10 can inhibit tumor cell growth in vitro. The inventor then further confirmed in a mouse model that CV-A10 has in vivo anti-tumor activity.

[0007] In order to study the pathogenic mechanism of enteroviruses such as CV-A10 and its possible anti-tumor mechanism, the inventors used cryo-electron microscopy to study the structure of the complex of CV-A10 and Kremen 1 (KRM1). In the process, the inventors also unexpectedly found that the CV-A10 binding site on KRM1 overlapped a lot with the binding site of DKK1 (Dickkopf-related protein 1) and KRM1, and then the inventors confirmed this binding site overlap in experiments. Based on the above findings, the inventors completed the present invention.

[0008] In one aspect, a method for inhibiting Wnt pathway signaling in a cell is provided, comprising exposing the cell to an enterovirus that uses KRM1 as a receptor, for example, the virus is selected from CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A10 and CV-A12, specifically CV-A10 virus.

[0009] Without being limited to any theory, the "enterovirus with KRM1 as a receptor" described herein completes the process of viral transmembrane invasion of host cells by binding to KRM1 as a viral receptor on the cell surface. This enterovirus is also referred to herein as an enterovirus with KRM1 as a receptor. For example, Coxsackievirus CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A10 and CV-A12 are mediated by KRM1 to achieve viral invasion and subsequent processes. They are all enteroviruses with KRM1 as a receptor described herein (Jacqueline Staring etc. KREMEN1 Is a Host Entry Receptor for a Major Group of Enteroviruses, 2018, Cell Host & Microbe 23, 636-643).

[0010] In a second aspect, a method for degrading β-catenin in a cell is provided, which comprises exposing the cell to an enterovirus that uses KRM1 as a receptor, for example, the virus is selected from CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A10 and CV-A12, specifically CV-A10 virus.

[0011] In one embodiment of the above aspects, the virus is engineered. For example, the engineered virus further comprises additional proteins and / or their coding sequences, and / or nucleic acids such as non-coding RNA that help achieve its purpose.

[0012] In a third aspect, a method for inhibiting tumor cell growth is provided, which comprises exposing the tumor cells to an enterovirus that uses KRM1 as a receptor, for example, the virus is selected from CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A10 and CV-A12, specifically CV-A10 virus.

[0013] In one embodiment, the tumor cells are, for example, cells from the following tumors: colon cancer, melanoma, prostate cancer, lung cancer such as non-small cell lung cancer, liver cancer, hepatocellular carcinoma, cervical cancer, uterine cancer, pancreatic cancer, gastric cancer, esophageal cancer, brain cancer, thyroblastoma and breast cancer.

[0014] In another embodiment, the virus is engineered. For example, the engineered virus also includes or is capable of producing additional proteins and / or their coding sequences and / or non-coding nucleic acids such as non-coding RNA that contribute to achieving its purpose, for example, in this aspect, the term "contributes to achieving its purpose" refers to contributing to inhibiting tumor cell growth. For example, the genome of the engineered virus also includes a nucleotide sequence encoding an additional anti-tumor agent or tumor therapeutic agent.

[0015] In a fourth aspect, a method for treating a tumor is provided, comprising administering to a subject in need thereof an enterovirus with KRM1 as a receptor, more preferably, the virus is selected from CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A10 and CV-A12, for example, CV-A10 virus.

[0016] In one embodiment, the tumor is selected from the group consisting of colon cancer, melanoma, prostate cancer, lung cancer such as non-small cell lung cancer, liver cancer, hepatocellular carcinoma, cervical cancer, uterine cancer, pancreatic cancer, gastric cancer, esophageal cancer, brain cancer, thyroblastoma and breast cancer.

[0017] In another embodiment, the virus is engineered. For example, this engineering can be any engineering modification that helps achieve its purpose. For example, in this aspect, the term "helps achieve its purpose" means helping to treat tumors. For example, it helps to store / transport viruses, helps to administer viruses, and helps to achieve better antiviral treatment effects. For example, the engineered virus also includes or can produce additional proteins, nucleic acids and / or their coding sequences that help achieve its purpose, and the nucleic acid is, for example, a non-coding nucleic acid such as a non-coding RNA. It is easy to understand that the term "engineering" herein can also include engineering modifications implemented to achieve other purposes.

[0018] In a specific embodiment, the genome of the engineered virus further comprises a nucleotide sequence encoding an additional tumor therapeutic agent. The additional tumor therapeutic agent may be, for example, a protein and / or nucleic acid such as a non-coding RNA for treating a tumor.

[0019] In another embodiment, the virus is administered to the subject by intratumoral injection, paracancer injection, intravenous injection and / or intramuscular injection. For example, the virus is administered by intratumoral injection, the virus is administered by paracancer injection, the virus is administered by intravenous injection, the virus is administered by intramuscular injection, the virus is administered by intratumoral injection and / or paracancer injection, the virus is administered by intratumoral injection and / or intravenous injection, and the virus is administered by intratumoral injection, paracancer injection and / or intravenous injection. Intratumoral injection is a typical administration route for anti-tumor viruses, and the virus can enter the treatment site directly without passing through the circulatory system. However, intratumoral injection often requires experienced professionals to implement. Paracancer injection is easier to operate, and professionals who have undergone preliminary training are usually competent. In paracancer injection, the therapeutic agent generally enters the treatment site slightly slower than intratumor injection, so paracancer injection is also relatively inferior to intratumor injection. However, in actual operation, the goal is that the operation of intratumoral injection is easy to inject near the cancer in practice. Intravenous injection is an operation that almost all medical professionals have mastered and does not require additional training. In intravenous injection, the therapeutic agent is carried into the treatment site with the circulatory system, and then the therapeutic agent will also be diluted by the circulatory system. Therefore, compared with intratumoral injection and para-cancer injection, intravenous injection is generally a secondary choice in anti-tumor virus therapy. The inventor unexpectedly found that in the use of the enterovirus of this article for anti-tumor treatment, intravenous injection has almost the same anti-tumor effect as intratumoral injection. Intramuscular injection has relatively lower requirements for auxiliary conditions, and it needs to be absorbed locally into the circulatory system, and then enter the site to be treated with the circulatory system.

[0020] In another embodiment, the virus is administered via a digestive tract route, such as oral administration. As is well known, the digestive tract route is not a common choice for anti-tumor viral therapy. The inventors unexpectedly discovered that even if the virus is administered via a digestive tract route, a significant anti-tumor effect can still be achieved.

[0021] In another embodiment, the method further comprises administering an additional anti-tumor therapy to the subject. The additional anti-tumor therapy is, for example, selected from one or more of chemotherapy, radiotherapy, targeted therapy, and immunotherapy.

[0022] It should be noted that although enterovirus is the pathogen of hand, foot and mouth disease, it is mainly caused by CV-A16 and EV-A71, and it is only more common in children under 5 years old to cause hand, foot and mouth disease. Most children have mild symptoms, and older children and adults rarely have significant symptoms. Based on the unexpected discovery of the present invention, this wide range of safety makes enterovirus with KRM1 as a receptor particularly suitable for treating tumors, especially tumors found in older children (greater than 5 years old) and adults. Therefore, in a specific embodiment, the treatment subject is an older child or an adult.

[0023] In a fifth aspect, an enterovirus with KRM1 as a receptor is provided, which is used to inhibit Wnt pathway signals in cells, to degrade β-catenin in cells, to inhibit the growth of tumor cells such as colon cancer cells, or to treat tumors such as colon cancer. For example, the virus is selected from CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A10 and CV-A12, specifically CV-A10 virus.

[0024] In one embodiment, the virus is engineered. For example, this engineering can be any genetic engineering modification that helps to achieve its purpose. For example, in this aspect, the term "helps to achieve its purpose" means to help treat tumors. For example, it helps to store / transport viruses, helps to administer viruses, and helps to achieve better antiviral treatment effects. For example, the engineered virus also includes or can produce additional proteins, nucleic acids and / or their coding sequences that help to achieve its purpose, and the nucleic acids are, for example, non-coding nucleic acids such as non-coding RNA. It is easy to understand that the term "engineering" herein can also include genetic engineering modifications implemented to achieve other purposes.

[0025] In a specific embodiment, the genome of the engineered virus further comprises a nucleotide sequence encoding an additional tumor therapeutic agent. The additional tumor therapeutic agent may be, for example, a protein and / or nucleic acid such as a non-coding RNA for treating a tumor.

[0026] In another embodiment, the virus is administered to the subject by intratumoral injection, paracancer injection, intravenous injection and / or intramuscular injection. For example, the virus is administered by intratumoral injection, the virus is administered by paracancer injection, the virus is administered by intravenous injection, the virus is administered by intramuscular injection, the virus is administered by intratumoral injection and / or paracancer injection, the virus is administered by intratumoral injection and / or intravenous injection, and the virus is administered by intratumoral injection, paracancer injection and / or intravenous injection. Intratumoral injection is a typical administration route for anti-tumor viruses, and the virus can enter the treatment site directly without passing through the circulatory system. However, intratumoral injection often requires experienced professionals to implement. Paracancer injection is easier to operate, and professionals who have undergone preliminary training are usually competent. In paracancer injection, the therapeutic agent generally enters the treatment site slightly slower than intratumor injection, so paracancer injection is also relatively inferior to intratumor injection. However, in actual operation, the goal is that the operation of intratumoral injection is easy to inject near the cancer in practice. Intravenous injection is an operation that almost all medical professionals have mastered and does not require additional training. In intravenous injection, the therapeutic agent is carried into the treatment site with the circulatory system, and then the therapeutic agent will also be diluted by the circulatory system. Therefore, compared with intratumoral injection and para-cancer injection, intravenous injection is generally a secondary choice in anti-tumor virus therapy. The inventor unexpectedly found that in the use of the enterovirus of this article for anti-tumor treatment, intravenous injection has almost the same anti-tumor effect as intratumoral injection. Intramuscular injection has relatively lower requirements for auxiliary conditions, and it needs to be absorbed locally into the circulatory system, and then enter the site to be treated with the circulatory system.

[0027] In another embodiment, the virus is administered via a digestive tract route, such as oral administration. As is well known, the digestive tract route is not a common choice for anti-tumor viral therapy. The inventors unexpectedly discovered that even if the virus is administered via a digestive tract route, a significant anti-tumor effect can still be achieved.

[0028] In the sixth aspect, a pharmaceutical composition is provided, comprising the enterovirus described in the sixth aspect, for inhibiting Wnt pathway signals in cells, for degrading β-catenin in cells, for inhibiting the growth of tumor cells such as colon cancer cells, or for treating tumors such as colon cancer, for example, the virus is selected from CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A10 and CV-A12, specifically CV-A10 virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The transmission electron micrograph of CV-A10 is shown. Scale bar: 100 nm.

[0030] Figure 2AIt was shown that CV-A10 inhibited the growth of human colon cancer cells RKO in vitro, and this inhibitory effect was positively correlated with the virus titer and gradually increased with time; Figure 2B EV-A71 infection of RKO cells is shown as a control.

[0031] Figure 3 shows tumor growth before and after injection of CV-A10 virus in NCG mice inoculated with RKO colon cancer cells. (A) Statistical results of intratumoral injection of CV-A10 virus, (B) Schematic diagram of tumor size after intratumoral injection, (C) Statistical results of intravenous injection of CV-A10 virus, (D) Schematic diagram of tumor size after intravenous injection.

[0032] Figure 4 The figure shows the purification and SDS-PAGE identification of the recombinant KRM1 protein.

[0033] Figure 5 Shown are the structure of the KRM1 and CV-A10 complex (A), the KRM1 and CV-A10 binding sites (B), and the KRM1 and DKK1 binding sites (C) structural comparison diagrams.

[0034] Figure 6 The relative expression levels of reporter genes in virus-infected cells are shown. CV-A10 inhibited the expression of downstream genes of the Wnt / β-catenin pathway in RD cells (A, B) and 293T cells (C, D), while EV-71, as a control, did not show a significant inhibitory effect, and DKK1 showed an inhibitory effect close to that of CV-A10.

[0035] Figure 7 The results showed that CV-A10 caused the degradation of β-catenin in RKO cells, and this effect was positively correlated with the viral load and increased with the increase of viral load.

[0036] Figure 8 Expression profiling of KRM1 in different stages of cancer (A) and in different tumors (B) is shown.

[0037] Fig. 9 It is shown that KRM1 expression is increased after CV-A10 infection of RKO cells.

[0038] FIG10 shows that CV-A10 inhibits the growth of human lung cancer cell lines MRC5, PC3, Calu-1 and NCI-h1299. This inhibitory effect is positively correlated with the virus titer and gradually increases with time. DETAILED DESCRIPTION

[0039] definition

[0040] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art. The following provides definitions of some terms used herein. If there are other interpretations of these terms, the definitions given herein shall prevail.

[0041] The term "virus" as used herein, such as "enterovirus", includes wild-type virus strains isolated directly from the environment or sick individuals, and also includes various virus variants derived from these wild-type viruses. On the other hand, in addition to the virus particles produced by conventional infection, the term "virus" also includes virus particles and virus-like particles (VLPs) produced by one or more processes such as artificial intervention in virus replication and assembly.

[0042] The term "engineered virus" as used herein refers to a virus or a variant thereof that has been genetically engineered and is different from a wild-type virus. Such genetic engineering may be for the purpose described herein or for other purposes.

[0043] The term "treatment" as used herein refers to any measure that is beneficial to the health status of the target subject, such as preventing the occurrence of disease, reducing the risk of disease occurrence, slowing down or hindering the progression of disease, inhibiting or stopping the growth of disease-related cells or entities (such as tumor cells or tumor entities), killing disease-related cells (such as tumor cells), shrinking disease-related entities (such as tumor entities), reducing or eliminating disease-related symptoms, preventing or slowing the occurrence or development of disease-related complications, inhibiting or slowing disease metastasis, and improving the survival rate of sick subjects.

[0044] The term "anti-tumor therapy" as used herein refers to any measure that helps prevent or delay tumor occurrence, reduce the risk of tumor occurrence, slow down or hinder tumor progression, inhibit or stop the growth of tumor cells or tumor entities, kill tumor cells, stop the growth or shrink tumor entities, reduce or eliminate tumor-related symptoms (such as tumor pain), prevent or slow the occurrence or development of tumor-related complications, inhibit or slow tumor metastasis, and improve the survival rate of patients. Anti-tumor therapy can be, for example, one or more of surgical resection, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. In medical practice, in order to slow down or eliminate the unhealthy state of patients, medical professionals often choose one or more applicable tumor treatment measures.

[0045] The terms "Wnt pathway" and "Wnt / β-catenin pathway" used herein refer to the signal transduction process mediated by Wnt and its receptor proteins. The Wnt pathway is an important pathway for intracellular signal transduction and is widely involved in a variety of physiological and pathological processes. The term "inhibiting Wnt pathway signals" refers to causing the Wnt pathway signals that mediate physiological processes to undergo changes that are opposite to the physiological processes, and is sometimes also referred to as negatively regulating Wnt pathway signals.

[0046] The term "selected from" as used herein means selected from a plurality of candidates. When applicable, in the absence of special indication, the "selected from" may be selected from a plurality of candidates.

[0047] Example

[0048] The embodiments of the present invention will be described in detail below in conjunction with examples. It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the examples, if specific conditions are not specified, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be purchased commercially.

[0049] Example 1: Amplification and purification of CV-A10 virus

[0050] Coxsackievirus A10 (CV-A10) HB09-035 strain was isolated in Hebei Province in 2009. The strain was deposited in China General Microbiological Culture Collection Center (No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China) with the deposit number CGMCC NO.19294 on January 19, 2020. Human rhabdomyosarcoma cells (RDcell, ATCC CCL-136) were cultured in DMEM medium containing 10% fetal bovine serum, 5×10 7 The cells were plated in 15 cm culture dishes and incubated at 37°C with 5% CO 2 Culture for 24 hours. Discard the culture medium and add CV-A10 virus at a multiplicity of infection (MOI) of 0.1. Dilute the virus with DMEM culture medium. Continue to culture for 36 hours. When 90% cytopathic effect (CPE) is observed under the microscope, collect the supernatant and use TFF systems 300kD concentrated virus and removed cell debris. Then ultracentrifuged at 140,000g for 2 hours and discarded the supernatant. Add PBS (pH7.4) to slowly resuspend the virus pellet and collect it in a microcentrifuge tube. Then pass the virus resuspension through a 15%-45% sucrose density gradient at 140,000g for 4 hours, collect each 200μl layer in separate tubes, and add an equal volume of PBS to reduce the osmotic pressure of the solution. Samples with OD260 / 280 greater than 1.5 were examined using a transmission electron microscope, and samples containing more mature virus particles (such as Figure 1 The virus was stored in PBS solution and the concentration and titer were determined before use.

[0051] Example 2: CV-A10 inhibits the growth of human colon cancer cell line RKO

[0052] Culture RKO colon cancer cell line (ATCC CRL-2577), take 2×10 4 Cells were placed in a 96-well plate and cultured for 8 hours. The culture medium was replaced with serum-free DMEM medium, infected with different titers of virus (MOI = 0.001-10), and continued to be cultured at 37°C. Cell viability was measured at different time points. The viability was measured using the CellTiter-Glo Luminescent CellViability Assay assays (Promega) kit according to the user manual. The results are shown in Figure 2A As shown, 12 hours after infection with a high virus titer (MOI=10), the cell survival rate began to decline, and almost all cells died after 48 hours. A lower virus titer (MOI=1) also caused almost all cancer cells to die after 60 hours. Other titer viruses produced gradient inhibitory effects at different times. The lowest virus titer (MOI=0.001) also showed inhibition of cell growth at 72 hours. This shows that CV-A10 can inhibit the growth of colon cancer cell line RKO in vitro, and is positively correlated with virus titer and infection time. Experiments were carried out with EV-A71 under the same conditions, and the results showed that EV-A71 had no significant growth inhibitory effect on human colon cancer cells RKO until 48 hours ( Figure 2B ), which shows that the effect of CV-A10 is specific.

[0053] Example 3: Intratumoral or intravenous injection of CV-A10 inhibits the growth of solid tumors in mice

[0054] A total of 15 4-week-old NCG mice (17-19 g) were selected and injected with 5×10 6 RKO cells / mouse. After tumor inoculation, the dorsal tumor was observed for 5 days. 3 The treatment was performed once every 3 days, that is, intratumoral or intravenous injection of CV-A10 virus was performed on days 5, 8, 11, 14, and 17. A control group was set up, with 5 mice in each group. Among them, the intratumoral injection group received multiple intratumoral injections, and the intravenous injection group received a single injection in the tail vein. The treatment dose was 1×10 7 Virus particles / time. The control group used PBS for multiple intratumoral injections to eliminate operational errors. The weight of the mice was recorded every two days, and the long and short diameters of the tumor were measured with a vernier caliper. The volume of the tumor was recorded and monitored continuously for 20 days. The mice were killed 20 days after tumor implantation, the tumors were removed, and the volume of the tumors was measured. From the tumor volume data, it can be seen that the tumor size of the intratumoral injection group was significantly different at 11 days (after 2 treatments, 6 days after treatment) and the intravenous injection group was at 13 days (after 3 treatments, 8 days after treatment). This proves that CV-A10 intratumoral injection ( Figure 3A-3B ) or intravenous injection ( Figure 3C-3D) can inhibit tumor growth.

[0055] Example 4: Expression and purification of receptor KRM1 protein

[0056] The inventors connected the DNA sequence encoding the extracellular segment of human Kremen 1 (KRM1) (amino acids 23-373) (Genbank number: AAH63787) into the pCMV3 vector (Beijing Sino Biological Technology Co., Ltd.) through the restriction enzyme sites HindIII and BamHI. The human type II interferon signal peptide was added to the 5' end of the KRM1 protein, and the coding sequence of the 10-histidine affinity tag (His10-tag) was added to the 3' end and a stop codon was added (the insertion sequence is shown in SEQ ID NO.1) to obtain the plasmid pCMV3-KRM1-ectodomain. 1×10 7 293T cells were placed in a 15 cm cell culture dish containing 20 ml of DMEM medium. When the cells grew to 70%, plasmid transfection was performed. 50 μg of plasmid and 150 μl of transfection reagent PEI (1 mg / ml) were mixed in buffer HBS (20 mM HEPES, 150 mM NaCl, pH 7.4). The transfection system was 2 ml in total. After mixing at room temperature for 30 minutes, it was added to the 15 cm culture dish where 293T cells were cultured. The supernatant was discarded 6 hours after transfection, and the culture medium was replaced with serum-free and transfection reagent-free DMEM culture medium, and the culture was continued for 3 days. After 3 days, the culture supernatant was collected and subjected to nickel ion affinity chromatography (HisTrap TM HP (GE)) and gel filtration chromatography (superdex TM 200increase 10 / 300GL(GE)) after purification, the protein purity was identified by SDS-PAGE, and the soluble human KRM1 extracellular segment protein (such as Figure 4 As shown). After identification, a high-purity KRM1 protein was obtained, with a size of about 70 kDa. The sequence encoding the extracellular region of recombinant human KRM1 is as follows:

[0057]

[0058] Example 5: The binding mode and action process of KRM1 and CV-A10 are similar to those of DKK1

[0059] The purified CV-A10 and receptor KRM1 in Examples 1 and 4 were incubated in vitro, and the purified CV-A10 virus (2 mg / ml) was incubated with an excess of KRM1 (0.2 mg / ml) for 5 minutes, and then the complex sample was adsorbed on a copper mesh with an ultra-thin carbon film (Lacey carbon, Zhongjing Keyi). After 1 minute of adsorption, the excess virus on the surface of the copper mesh was blotted dry with filter paper, and the sample was quickly inserted into liquid ethane with the help of a Vitrobot Mark IV (FEI) freezing sample preparation machine, and then transferred to liquid nitrogen to preserve the sample. Data collection was completed using a 200kV Arctica (FEI) electron microscope equipped with a Gatan K2 direct electron detector. All original electron microscope photos were drift corrected using the MotionCor2 program. The contrast transfer function (CTF) correction was calculated using CTFFIND4, and EMAN2 was used for automatic particle selection. Finally, the resolution was calculated using Relion. The final resolution was estimated by the Fourier shell correlation function (FSC) curve. By simulating the neutral environment of extracellular pH 7.4 under physiological conditions, it was confirmed that KRM1 binds to the CV-A10 virus through its Kringle (KR) and WSC domains, and its binding mode is basically consistent with the site where KRM1 binds to DKK1 in the Wnt / β-catenin signaling pathway. Figure 5 As shown, by comparing the binding modes of KRM1 and DKK1 (PDB code: 5FWW), it was shown that CV-A10 can mimic DKK1 to inhibit the Wnt / β-catenin signaling pathway.

[0060] Example 6: CV-A10 inhibits Wnt / β-catenin pathway signal transduction

[0061] In this example, EV-A71 virus was purified as a control group by a method similar to that of amplifying and purifying CV-A10 in Example 1. A full-length plasmid of KRM1 was constructed by a method similar to that of Example 4, i.e., the amino acid coding sequence at positions 1-473 (Genbank number: AAH63787) was connected to the vector pLVX-DsRed-Monomer-N1 (Clontech) through the 5'XhoI and 3'NotI restriction enzyme sites, and a Flag tag and a stop codon were added at the 3' end, and KRM1 was overexpressed in cells by exogenous transfection.

[0062] 293T or RD cells were cultured in a 24-well cell culture plate, and the reporter gene plasmid Top-Flash (Beyotime, D2501) and the internal control pRenilla-TK plasmid were transfected into the cells. The constructed full-length KRM1 overexpression plasmid was added to one group at the same time. The transfection system was: 3 replicate wells per group, 90μl HBS solution, 70μl PEI (0.1mg / ml) and 2μg plasmid were prepared. The DMEM medium was replaced 6 hours after transfection. 12 hours after transfection, Wnt3a (R&D, 1324-WN-500 / CF) was added to the culture medium, and the cells were infected with CV-A10 or EV-A71 virus (MOI=1), respectively. After 24 hours of treatment, the cells were lysed for luciferase activity detection. Figure 6 As shown in Figure 2, CV-A10 has a significant inhibitory effect on the Wnt / β-catenin signaling pathway, and this effect is enhanced when KRM1 is overexpressed ( Figure 6 B, 6D), basically blocking the activation of Wnt3a on the signal. However, EV-A71 virus has no effect on the expression of the reporter gene ( Figure 6 A, 6B). Therefore, CV-A10 can act as an inhibitor of the Wnt / β-catenin pathway. In addition, when DKK1 protein (50 ng / ml, purchased from Beijing Sino Biological Technology Co., Ltd.) was added to the 293T group, both DKK1 and CV-A10 showed inhibitory effects on the Wnt / β-catenin signaling pathway, while the inhibitory effect did not double when both were added at the same time ( Figure 6 C, 6D). This result confirmed that CV-A10 inhibited the Wnt / β-catenin signaling pathway through the viral receptor KRM1. The binding epitope of CV-A10 and KRM1 was similar to that of DKK1, and there was competitive binding between the two.

[0063] Example 7: CV-A10 causes degradation of β-catenin and inhibits the Wnt / β-catenin pathway

[0064] RKO colon cancer cells 1×10 6Cultured in 6-well plates, 12 hours later, when the cells grew to 90%, they were infected with CV-A10 (MOI=1) and viruses of different titers (MOI=0.001-10), and continued to be cultured at 37°C. RKO cells infected with CV-A10 (MOI=1) were washed twice with PBS at 36 hours, lysed with IP lysis buffer, and then analyzed by Western blot. Cells infected with different titers of viruses (MOI=0.001-10) were washed twice with PBS at 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, and 72 hours, lysed with 200 μl IP lysis buffer, and then analyzed by Western blot. Figure 7 As shown in Figure 2, the expression of β-catenin in RKO cells decreased with the extension of infection time ( Figure 7 A); Under the same infection time, β-catenin decreases with the increase of added virus amount ( Figure 7 B). This indicates that CV-A10 inhibits the Wnt / β-catenin pathway through the degradation of β-catenin. Similar results were obtained by infecting 293T cells with CV-A10 ( Figure 7 C).

[0065] Example 8: Tissue expression profile of KRM1, a key receptor for CV-A10 invasion

[0066] Using The Cancer Genome Atlas (TCGA) database, we found that there was no significant difference in the expression level of KRM1 at different stages of cancer infection ( Figure 8 A). However, it is worth noting that the expression of KRM1 varies greatly in different tumors. It is highly expressed in some tumors such as renal chromophobe cell carcinoma (KICH), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and renal clear cell carcinoma (KIRC). In particular, the expression level in colon cancer is significantly higher than that in normal tissues ( Figure 8 B). This suggests that CV-A10 may have a more significant therapeutic effect on these tumors.

[0067] In vitro immunofluorescence experiments further revealed that KRM1 expression in RKO cells increased after infection with CV-A10 ( Fig. 9 ). This indicates that CV-A10 infection of cells causes an increase in KRM1 expression in cells, which will lead to infection by more viruses, thus playing a positive feedback role.

[0068] Example 9: CV-A10 reduces the survival rate of lung cancer cells

[0069] MRC5 (ATCC, CCL-171), PC3 (ATCC, CRL-1435), Calu-1 (ATCC, HTB-54), and NCI-h1299 (ATCC, CRL-5083) lung cancer cell lines were cultured, 2×10 4 Cells were plated in 96-well plates. After 8 h of culture, the medium was changed to serum-free DMEM medium, infected with different titers of virus (MOI = 0.001-10), and continued to be cultured at 37°C. Cell viability was measured at different time points using the CellTiter-GloLuminescent Cell Viability Assay assays (Promega). Figures 10A-10D As shown in the figure, 4 lung cancer cells infected with high virus titer (MOI=10) began to show a trend of decreasing survival rate 12 hours after infection, and almost all died after 48 hours. A higher virus titer (MOI=1) caused almost all cancer cells to die after 60 hours. Other titer viruses produced gradient inhibitory effects at different times. The lowest virus titer (MOI=0.001) also showed inhibition of cell growth at 72 hours. This shows that CV-A10 can cause a decrease in the survival rate of lung cancer cells, and its effect is positively correlated with the virus titer and infection time. Sequence Listing <110> Shanxi Advanced Innovation Institute <120> An anti-tumor virus <130> MP2031367 <160> 1 <170> PatentIn version 3.3 <210> 1 <211> 1239 <212> DNA <213> Artificial sequence <220> <223> Recombinant human KRM1 extracellular domain coding sequence <400> 1 aagcttgcca ccatgggcat tctgcccagc cccggcatgc ccgctctgct gtctctggtg 60 tctctgctga gcgtgctgct gatgggctgc gtggctgaga ccggagctcc ttcccccgga 120 ctgggacccg gacccgagtg ctttaccgcc aacggcgccg actacagagg aacacagaat 180 tggaccgctc tgcaaggagg aaagccttgt ctgttctgga acgagacatt ccaacacccc 240 tacaacaccc tcaagtaccc caacggagag ggaggactgg gagaacacaa ctactgcaga 300 aaccccgacg gcgatgtgag cccttggtgc tacgtcgccg aacacgagga cggagtctac 360 tggaagtact gcgaaatccc cgcttgccaa atgcccggca atctgggctg ctacaaggat 420 cacggaaacc cccctcccct caccggcacc tccaagacct ccaacaagct caccatccag 480 acatgcatca gcttctgcag atcccagagg tttaagttcg ccggcatgga gtccggctat 540 gcttgcttct gcggcaacaa ccccgactat tggaagtacg gcgaagctgc cagcaccgag 600 tgcaattccg tgtgcttcgg cgatcacacc cagccttgcg gaggagacgg aagaatcatt 660 ctgtttgaca cactggtggg cgcttgcggc ggaaactact ccgccatgag cagcgtggtg 720 tacagccccg acttccccga cacctacgcc accggcagag tgtgttactg gaccattaga 780 gtgcccggcg ccagccacat ccactttagc ttccctctgt tcgacattag agatagcgct 840 gacatggtcg agctgctgga tggatacacc catagggtgc tggctagatt ccacggaagg 900 agcagacctc ctctgtcctt caacgtctct ctggacttcg tgattctgta cttcttcagc 960 gatagaatca accaagccca aggcttcgcc gtcctctatc aagccgtgaa agaggagggc 1020 agcgagaacc tctactttca aggcggatct ctgccccaag agagacccgc cgtcaaccaa 1080 acagtggccg aggtgattac agagcaagcc aatctgagcg tgtccgctgc tagaagctcc 1140 aaggtgctgt atgtgatcac cacctcccct agccatcccc cccagacagt gcccggcaca 1200 caccaccacc accatcacca ccatcatcac tgaggatcc 1239

Claims

1. A method for inhibiting Wnt pathway signaling in cells in vitro, comprising exposing the cells to an enterovirus that uses KRM1 as a receptor, wherein the virus is CV-A10.

2. The method according to claim 1, wherein the virus was deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO.19294 on January 19, 2020.

3. A method for degrading β-catenin in cells in vitro, comprising exposing the cells to an enterovirus that uses KRM1 as a receptor, wherein the virus is CV-A10.

4. The method according to claim 3, wherein the virus was deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO.19294 on January 19, 2020.

5. A method for inhibiting tumor cell growth in vitro, comprising exposing the tumor cell to an enterovirus that uses KRM1 as a receptor, wherein the virus is CV-A10, and the tumor cell is a colon cancer cell or a lung cancer cell.

6. The method according to claim 5, wherein the virus was deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO.19294 on January 19, 2020.

7. Use of an enterovirus with KRM1 as a receptor in the preparation of a drug for treating tumors, wherein the virus is CV-A10 and the tumor is colon cancer or lung cancer.

8. The use according to claim 7, wherein the virus was deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO.19294 on January 19, 2020.

9. The use according to claim 7, wherein the virus is engineered.

10. The use according to claim 9, wherein the genome of the engineered virus further comprises a nucleotide sequence encoding an additional tumor therapeutic agent.

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