WO2021147874A1 - 一种抗肿瘤病毒 - Google Patents

一种抗肿瘤病毒 Download PDF

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WO2021147874A1
WO2021147874A1 PCT/CN2021/072775 CN2021072775W WO2021147874A1 WO 2021147874 A1 WO2021147874 A1 WO 2021147874A1 CN 2021072775 W CN2021072775 W CN 2021072775W WO 2021147874 A1 WO2021147874 A1 WO 2021147874A1
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virus
cells
tumor
krm1
injection
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French (fr)
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高山
崔樱子
仝舟
高福
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山西高等创新研究院
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Priority to US17/794,217 priority Critical patent/US20230057747A1/en
Priority to EP21743917.3A priority patent/EP4095241A4/en
Priority to JP2022569290A priority patent/JP7421244B2/ja
Publication of WO2021147874A1 publication Critical patent/WO2021147874A1/zh

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Definitions

  • the present invention relates to a virus that inhibits Wnt signal and a method of using the virus to inhibit Wnt signal, and also relates to a method of using the virus to treat tumors.
  • Hand, foot and mouth disease is an infectious disease that occurs mostly in children under 5 years of age and is rare in adults. Pathways of infection include 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 herpes or small ulcers in the hands, feet, and mouth. Most children heal by themselves within a week, and a few cause complications such as myocarditis, pulmonary edema, and aseptic meningoencephalitis. Some severe illnesses develop rapidly. , Leading to death. There is no effective treatment drug, mainly symptomatic treatment.
  • enteroviruses can cause hand, foot and mouth disease.
  • the most common pathogens are Coxsackie virus A16 (CV-A16, also known as CVA16) and enterovirus 71 (EV-A71, also known as EV-71 or EV71), they all belong to the Enterovirus genus of the Picornaviridae.
  • CV-A16 Coxsackie virus A16
  • EV-A71 enterovirus 71
  • EV-71 also known as EV-71 or EV71
  • Coxsackie virus is divided into two types: A (type 23) and B (type 6). Although CV-A16 is an important pathogen of hand, foot and mouth disease, 90% of Coxsackie virus infections cause no symptoms or only fever.
  • CV-A10 Coxsackie virus A10
  • CVA10 Coxsackie virus A10
  • the inventors used cryo-electron microscopy to study the structure of the complex of CV-A10 and Kremen 1 (KRM1). During this process, the inventor also unexpectedly discovered that the CV-A10 binding site on KRM1 overlaps with the binding site of DKK1 (Dickkopf-related protein 1) and KRM1. Subsequently, the inventors confirmed this binding in experiments. The sites overlap. Based on the above findings, the inventor completed the present invention.
  • a method for inhibiting Wnt pathway signaling in a cell 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.
  • the "enteric virus with KRM1 as the receptor” described herein completes the process of virus transmembrane invasion of host cells by binding to KRM1 as the virus receptor on the cell surface.
  • this enterovirus is also referred to as an enterovirus with KRM1 as the receptor.
  • the Coxsackie virus CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A10 and CV-A12 are mediated by KRM1 to achieve virus invasion and subsequent processes. They are all mentioned in this article.
  • the aforementioned enterovirus with KRM1 as the receptor Jacqueline Staring etc.
  • KREMEN1 Is a Host Entry Receptor for a Major Group of Enteroviruses, 2018, Cell Host & Microbe 23,636-643).
  • a method for degrading ⁇ -catenin in a cell 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.
  • the virus is engineered.
  • the engineered virus also contains additional protein and/or its coding sequence, and/or nucleic acid such as non-coding RNA that help achieve its purpose.
  • a method for inhibiting the growth of tumor cells 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.
  • 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, thyroid cell carcinoma and breast cancer.
  • the virus is engineered.
  • engineered viruses also include or are capable of producing additional proteins and/or their coding sequences, and/or non-coding nucleic acids such as non-coding RNAs that help achieve their purpose.
  • help achieve "The purpose” means to help inhibit the growth of tumor cells.
  • the genome of the engineered virus also includes nucleotide sequences encoding additional anti-tumor agents or tumor therapeutic agents.
  • a method for treating tumors comprises administering to a subject in need 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, for example CV-A10 virus.
  • the tumor is selected from: colon cancer, melanoma, prostate cancer, lung cancer such as non-small cell lung cancer, liver cancer, hepatocellular carcinoma, cervical cancer, uterine cancer, pancreatic cancer, stomach cancer, esophageal cancer, brain Cancer, thyroid cell carcinoma and breast cancer.
  • lung cancer such as non-small cell lung cancer, liver cancer, hepatocellular carcinoma, cervical cancer, uterine cancer, pancreatic cancer, stomach cancer, esophageal cancer, brain Cancer, thyroid cell carcinoma and breast cancer.
  • the virus is engineered.
  • this engineering can be any engineering that helps to achieve its purpose.
  • help to achieve its purpose means to help treat tumors.
  • an engineered virus also includes or is capable of producing additional proteins, nucleic acids, and/or coding sequences thereof that help achieve its purpose, such as non-coding nucleic acids such as non-coding RNA. It is easy to understand that the term "engineering" herein can also include engineering modifications implemented to achieve other purposes.
  • the genome of the engineered virus further includes a nucleotide sequence encoding an additional tumor therapeutic agent.
  • the additional tumor therapeutic agent may be a protein and/or nucleic acid such as non-coding RNA to treat tumors.
  • the virus is administered to the subject by intratumoral injection, paracancerous injection, intravenous injection, and/or intramuscular injection.
  • the virus is administered by intratumoral injection, the virus is administered by paracancerous injection, the virus is administered by intravenous injection, the virus is administered by intramuscular injection, the virus is administered by intratumoral injection and/or paracancerous injection, The virus is administered by intratumor injection and/or intravenous injection, and the virus is administered by intratumor injection, paracancerous injection, and/or intravenous injection.
  • Intratumoral injection is a typical route of administration of anti-tumor viruses, and the virus can directly enter the treatment site without passing through the circulatory system. However, intratumoral injections often require experienced professionals to implement.
  • Intravenous injection is an operation that is mastered by almost all medical professionals and does not require additional training.
  • intravenous injection the therapeutic agent is brought into the treatment site along with the circulatory system, and the therapeutic agent will also be diluted by the circulatory system. Therefore, compared with intratumor injection and paracancerous injection, intravenous injection is generally the second choice in anti-tumor virus therapy.
  • Intramuscular injection has relatively lower requirements for auxiliary conditions. It needs to be absorbed locally into the circulatory system, and then enter the site to be treated with the circulatory system.
  • the virus is administered via the digestive tract route, such as orally.
  • the digestive tract route is not the usual choice for anti-tumor viral therapy. The inventors unexpectedly discovered that even if the virus is administered through the digestive tract route, significant anti-tumor effects can still be achieved.
  • the method further comprises administering 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.
  • enterovirus is the pathogen of hand, foot and mouth disease, it is mainly caused by CV-A16 and EV-A71, and it is more common in children under 5 years of age to cause hand, foot and mouth disease. Most children have mild symptoms. Older children and adults rarely see significant symptoms. Based on the unexpected discovery of the present invention, this wide range of safety makes enteroviruses using KRM1 as the receptor particularly suitable for the treatment of tumors, especially tumors found in older children (greater than 5 years old) and adults. Therefore, in a specific embodiment, the subject of treatment is an older child or adult.
  • an enterovirus with KRM1 as the receptor is provided, which is used to inhibit Wnt pathway signals in cells, to degrade ⁇ -catenin in cells, and to inhibit the growth of tumor cells such as colon cancer cells.
  • the virus is selected from CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A10 and CV-A12, specifically CV-A10 Virus.
  • the virus is engineered.
  • this engineering can be any genetic engineering that helps to achieve its purpose.
  • help to achieve its purpose means to help treat tumors.
  • an engineered virus also includes or is capable of producing additional proteins, nucleic acids, and/or coding sequences thereof that help achieve its purpose, such as non-coding nucleic acids such as non-coding RNA. It is easy to understand that the term "engineered” herein can also include genetic engineering implemented for other purposes.
  • the genome of the engineered virus further includes a nucleotide sequence encoding an additional tumor therapeutic agent.
  • the additional tumor therapeutic agent may be a protein and/or nucleic acid such as non-coding RNA to treat tumors.
  • the virus is administered to the subject by intratumoral injection, paracancerous injection, intravenous injection, and/or intramuscular injection.
  • the virus is administered by intratumoral injection, the virus is administered by paracancerous injection, the virus is administered by intravenous injection, the virus is administered by intramuscular injection, the virus is administered by intratumoral injection and/or paracancerous injection, The virus is administered by intratumor injection and/or intravenous injection, and the virus is administered by intratumor injection, paracancerous injection, and/or intravenous injection.
  • Intratumoral injection is a typical route of administration of anti-tumor viruses, and the virus can directly enter the treatment site without passing through the circulatory system. However, intratumoral injections often require experienced professionals to implement.
  • Intravenous injection is an operation that is mastered by almost all medical professionals and does not require additional training.
  • intravenous injection the therapeutic agent is brought into the treatment site along with the circulatory system, and the therapeutic agent will also be diluted by the circulatory system. Therefore, compared with intratumor injection and paracancerous injection, intravenous injection is generally the second choice in anti-tumor virus therapy.
  • Intramuscular injection has relatively lower requirements for auxiliary conditions. It needs to be absorbed locally into the circulatory system, and then enter the site to be treated with the circulatory system.
  • the virus is administered via the digestive tract route, such as orally.
  • the digestive tract route is not the usual choice for anti-tumor viral therapy. The inventors unexpectedly discovered that even if the virus is administered via the digestive tract route, significant anti-tumor effects can still be achieved.
  • a pharmaceutical composition comprising the enterovirus described in the sixth aspect, for inhibiting Wnt pathway signals in cells, for degrading ⁇ -catenin in cells, and for inhibiting Growth of tumor cells such as colon cancer cells, or for the treatment of 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 the CV-A10 virus.
  • Figure 1 shows a transmission electron microscope image of CV-A10. Scale bar: 100nm.
  • Figure 2A shows that CV-A10 inhibits the growth of human colon cancer cells RKO in vitro. This inhibitory effect is positively correlated with virus titer and gradually increases with time;
  • Figure 2B shows EV-A71 infection of RKO cells as a control.
  • Figure 3 shows tumor growth before and after injection of CV-A10 virus in NCG mice inoculated with RKO colon cancer cells.
  • A The statistical results of intratumoral injection of CV-A10 virus
  • B the schematic diagram of tumor size after intratumoral injection
  • C the statistical results of intravenous injection of CV-A10 virus
  • D the schematic diagram of tumor size after intravenous injection.
  • Figure 4 shows the purification and SDS-PAGE identification of recombinant KRM1 protein.
  • FIG 5 shows the structure of the KRM1 and CV-A10 complex (A), the binding site of KRM1 and CV-A10 (B), and the comparison of the structure of the binding site of KRM1 and DKK1 (C).
  • Figure 6 shows the relative expression levels of reporter genes in virus-infected cells.
  • CV-A10 inhibits the downstream gene expression of Wnt/ ⁇ -catenin pathway signaling in RD cells (A, B) and 293T cells (C, D).
  • RD cells A, B
  • C 293T cells
  • EV-71 does not show significant inhibition.
  • DKK1 showed an inhibitory effect close to that of CV-A10.
  • Figure 7 shows that CV-A10 leads to the degradation of ⁇ -catenin in RKO cells. This effect is positively correlated with the amount of virus and increases with the increase of virus amount.
  • Figure 8 shows the expression profile analysis of KRM1 in different stages of cancer (A) and different tumors (B).
  • Figure 9 shows the increase in KRM1 expression after CV-A10 infection of RKO cells.
  • Figure 10 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 virus titer and gradually increases with time.
  • virus such as "enterovirus” includes wild-type virus strains directly isolated from the environment or diseased individuals, as well as various virus variants derived from these wild-type viruses.
  • virus particles produced by conventional infections
  • virus particles also includes virus particles and virus-like particles (VLP) produced by human intervention in one or more processes such as virus replication and assembly.
  • VLP virus-like particles
  • engineered virus refers to a virus that is genetically engineered or a variant thereof that is different from the wild-type virus. This genetic engineering can be performed for the purpose described herein or for other purposes. Genetic engineering.
  • treatment refers to any measure that is beneficial to the health of the target object, for example, it can prevent the occurrence of disease, reduce the risk of disease occurrence, slow down or hinder the disease process, inhibit or stop disease-related cells or entities (such as tumor cells or tumors).
  • the growth of entities kill disease-related cells (such as tumor cells), shrink disease-related entities (such as tumor entities), reduce or eliminate disease-related symptoms, prevent or slow down the occurrence or development of disease-related complications, and inhibit or slow down
  • the disease transfers and improves the survival rate of sick subjects.
  • anti-tumor therapy refers to help prevent or delay tumor occurrence, reduce the risk of tumor occurrence, slow or hinder tumor progression, inhibit or stop the growth of tumor cells or tumor entities, kill tumor cells, and stop tumor entities Any measures to grow or shrink, reduce or eliminate tumor-related symptoms (such as tumor pain), prevent or slow down the occurrence or development of tumor-related complications, inhibit or slow down tumor metastasis, and improve the survival rate of patients.
  • the anti-tumor therapy may 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 the sick subject, medical professionals often choose one or more applicable tumor treatment measures.
  • Wnt pathway and “Wnt/ ⁇ -catenin pathway” as used herein refer to the signal transmission process mediated by Wnt and its receptor protein. Wnt pathway is an important pathway of intracellular signal transmission, which is widely involved in a variety of physiological and pathological processes.
  • inhibitor of Wnt pathway signals refers to making Wnt pathway signals that mediate physiological processes change opposite to physiological processes, and is sometimes referred to as negatively regulating Wnt pathway signals.
  • selected from refers to selecting from multiple candidates. When applicable, unless otherwise specified, the “selected from” may be to select one or more candidates from a plurality of candidates.
  • Example 1 Amplification and purification of CV-A10 virus
  • Coxsackie virus A10 (CV-A10) HB09-035 strain was isolated in Hebeizhou in 2009. The virus strain was deposited in the China General Microbial Culture Collection Management Center (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China) under the deposit number CGMCC NO.19294 on January 19, 2020.
  • Human rhabdomyosarcoma cells (RD cell, ATCC CCL-136) were cultured in DMEM medium containing 10% fetal bovine serum, 5 ⁇ 10 7 cells were seeded in a 15 cm culture dish, 37°C with 5% CO 2 Incubate for 24 hours.
  • DMEM medium fetal calf serum
  • the virus resuspension was centrifuged at 140,000 g through a 15%-45% sucrose density gradient for 4 hours, with 200 ⁇ l as a layer, collected in separate tubes, and an equal volume of PBS was added to reduce the osmotic pressure of the solution.
  • the samples with OD260/280 greater than 1.5 are examined by transmission electron microscopy, and samples with more mature virus particles are retained (as shown in Figure 1).
  • the virus was stored in PBS solution, and the concentration and titer were determined for later use.
  • Example 2 CV-A10 inhibits the growth of human colon cancer cell line RKO
  • the RKO colon cancer cell line (ATCC CRL-2577) was cultured, and 2 ⁇ 10 4 cells were placed in a 96-well plate and cultured for 8 hours.
  • Example 3 Intratumor or intravenous injection of CV-A10 inhibits the growth of solid tumors in mice
  • a total of 15 4-week-old NCG mice (17-19g) were selected and injected with 5 ⁇ 10 6 RKO cells/mouse on the back. After planting the tumors, they were reared for 5 days to observe the tumor formation on the back.
  • the tumor grows to 120 mm 3 treatment is performed every 3 days, that is, intratumoral injection or intravenous injection of CV-A10 virus is performed on days 5, 8, 11, 14, and 17, and a control group is set up with 5 mice in each group.
  • the intratumoral injection group was intratumoral multi-point injection
  • the intravenous injection group was tail vein single-needle injection
  • the therapeutic dose was 1 ⁇ 10 7 virus particles/time.
  • PBS was used for intratumoral multi-point injection to eliminate operation errors.
  • mice The body weight of the mice was recorded every two days and the long and short diameters of the tumor were measured with a vernier caliper, and the volume of the tumor was recorded, and the monitoring was continued for 20 days.
  • the mice were sacrificed 20 days after the tumor was implanted, the tumor was taken out, and the tumor volume was measured. From the tumor volume data, it can be seen that the tumor size has become significant on the 11th day in the intratumoral injection group (after 2 treatments, 6 days after treatment) and the intravenous injection group on the 13th day (after 3 times treatment, 8 days after treatment). difference. It is proved that CV-A10 intratumor injection (Figure 3A-3B) or intravenous injection (Figure 3C-3D) can inhibit tumor growth.
  • the inventors ligated the DNA sequence (Genbank number: AAH63787) encoding the extracellular segment of human Kremen 1 (KRM1) (amino acids 23-373) into the pCMV3 vector (Beijing Yiqiao Shenzhou Technology Co., Ltd.) through restriction enzyme cleavage sites HindIII and BamHI Limited company).
  • KRM1 extracellular segment of human Kremen 1
  • pCMV3 vector Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • HindIII and BamHI Limited company restriction enzyme cleavage sites HindIII and BamHI Limited company.
  • the 5'end of the KRM1 protein was added with a human type II interferon signal peptide, and a 10 histidine affinity tag (His10-tag) coding sequence was added at the 3'end, and a stop codon was added (the insert sequence is as SEQ ID NO .1), the plasmid pCMV3-KRM1-ectodomain was
  • the sequence encoding the extracellular region of recombinant human KRM1 is as follows:
  • Example 5 The binding mode and action process of KRM1 and CV-A10 are similar to DKK1
  • the CV-A10 purified in Examples 1 and 4 and the receptor KRM1 were incubated in vitro, and the purified CV-A10 virus (2mg/ml) was incubated with excess KRM1 (0.2mg/ml) for 5 minutes, and then the complex was incubated
  • the sample is adsorbed on a copper mesh (Lacey carbon) with an ultra-thin carbon film. After adsorbing for 1 minute, the excess virus on the surface of the copper mesh is blotted dry with filter paper, and the Vitrobot Mark IV (FEI) freezing sample preparation machine is used. Quickly insert it into liquid ethane, and then transfer it to liquid nitrogen to store the sample.
  • FEI Vitrobot Mark IV
  • KRM1 binds to CV-A10 virus through its Kringle (KR) and WSC domains, and its binding mode is the same as that of KRM1 binding to DKK1 in the Wnt/ ⁇ -catenin signaling pathway.
  • the sites are basically the same.
  • KRM1 and DKK1 (PDB code: 5FWW)
  • CV-A10 can mimic DKK1 and inhibit the Wnt/ ⁇ -catenin signaling pathway.
  • Example 6 CV-A10 inhibits Wnt/ ⁇ -catenin pathway signal transduction
  • the EV-A71 virus was purified using a method similar to the amplification and purification of CV-A10 in Example 1 as a control group. And refer to the similar method of Example 4 to construct the KRM1 full-length plasmid, that is, the amino acid coding sequence at positions 1-473 (Genbank number: AAH63787) is ligated into the vector pLVX-DsRed-Monomer through 5'XhoI and 3'NotI restriction enzyme sites -N1 (Clontech), and add a Flag tag and a stop codon at the 3'end to overexpress KRM1 through exogenous transfection.
  • the KRM1 full-length plasmid that is, the amino acid coding sequence at positions 1-473 (Genbank number: AAH63787) is ligated into the vector pLVX-DsRed-Monomer through 5'XhoI and 3'NotI restriction enzyme sites -N1 (Clontech), and add a Flag tag and a stop codon
  • CV-A10 can act as a Wnt/ ⁇ -catenin pathway inhibitor.
  • DKK1 protein 50ng/ml, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • Both DKK1 and CV-A10 showed inhibitory effects on the Wnt/ ⁇ -catenin signaling pathway, and both were added at the same time At this time, its inhibitory effect did not double ( Figure 6C, 6D). It was confirmed once again that CV-A10 inhibits the Wnt/ ⁇ -catenin signaling pathway through the viral receptor KRM1.
  • the binding epitope of CV-A10 and KRM1 is similar to that of DKK1, and there is a competitive binding between the two.
  • Example 7 CV-A10 leads to the degradation of ⁇ -catenin and thereby inhibits the Wnt/ ⁇ -catenin pathway
  • Example 8 Tissue expression profile of CV-A10 invasion key receptor KRM1
  • KICH renal chromophobe cell carcinoma
  • LAD lung adenocarcinoma
  • LUSC lung squamous cell carcinoma
  • KIRC renal clear cell carcinoma
  • Example 9 CV-A10 reduces the survival rate of lung cancer cells
  • Viruses of other titers produce gradient inhibitory effects at different times.

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Abstract

公开了一种抑制Wnt信号的病毒以及使用所述病毒抑制Wnt信号的方法，还公开了一种使用所述病毒治疗肿瘤的方法。

Description

一种抗肿瘤病毒
本申请要求于2020年01月20日提交中国专利局、申请号为202010065206.8的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及一种抑制Wnt信号的病毒以及使用所述病毒抑制Wnt信号的方法，还涉及使用所述病毒治疗肿瘤的方法。
背景技术
手足口病是一种多发于5岁以下幼儿的传染病，成人很少见。感染途径包括消化道、呼吸道和接触传播。手足口病的潜伏期2-10天，平均3-5天。症状表现为口痛、厌食、低热、手足口等部位出现小疱疹或小溃疡，多数患儿一周自愈，少数引起心肌炎、肺水肿、无菌性脑膜脑炎等并发症，个别重症病情发展快，导致死亡。尚无有效治疗药物，主要对症治疗。
多种肠道病毒可引起手足口病，最常见的致病原是柯萨奇病毒A16型(CV-A16，亦称CVA16)和肠道病毒71型(EV-A71，亦称EV-71或EV71)，它们都属于小RNA病毒科肠病毒属。
柯萨奇病毒分为A(23型)和B(6型)两类。尽管CV-A16是手足口病的重要致病原，90％的柯萨奇病毒感染不引起症状或仅有发热。
迄今人们对这些肠道病毒的了解仍然很有限，因此有必要进一步研究肠道病毒的致病谱以及发病机理。
发明内容
在以柯萨奇病毒A10型(CV-A10，亦称CVA10)为模型研究肠道病毒的致病谱的过程中，发明人意外地发现，肠道病毒如CV-A10能够在体 外抑制肿瘤细胞生长。发明人随后在小鼠模型中进一步证实了CV-A10具有体内抗肿瘤活性。
为了研究肠道病毒如CV-A10致病机理以及其可能的抗肿瘤机制，发明人使用冷冻电镜技术研究了CV-A10与Kremen 1(KRM1)复合物的结构。在此过程中发明人还意外地发现，KRM1上的CV-A10结合位点与DKK1(Dickkopf-related protein 1)与KRM1的结合位点有大量重叠，随后发明人在实验中证实了这种结合位点重叠。在以上发现的基础上，发明人完成了本发明。
在一个方面中，提供一种抑制细胞中Wnt通路信号的方法，其包括使所述细胞暴露于以KRM1为受体的肠道病毒，例如所述病毒选自CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12，具体地是CV-A10病毒。
不限于任何理论，本文所述“以KRM1为受体的肠道病毒”通过与细胞表面作为病毒受体的KRM1结合从而完成病毒跨膜入侵宿主细胞的过程。本文中又称这种肠道病毒为以KRM1为受体的肠道病毒。例如，柯萨奇病毒CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12通过KRM1介导来实现病毒入侵和后续过程，它们都是本文所述的以KRM1为受体的肠道病毒(Jacqueline Staring etc.KREMEN1 Is a Host Entry Receptor for a Major Group of Enteroviruses,2018,Cell Host&Microbe 23,636-643)。
在第二个方面中，提供一种使细胞中β-catenin降解的方法，其包括使所述细胞暴露于以KRM1为受体的肠道病毒，例如所述病毒选自CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12，具体地是CV-A10病毒。
在上述方面的一个实施方案中，所述病毒是工程化的。例如工程化的病毒还包含有助于实现其目的的额外的蛋白质和/或其编码序列、和/或核酸如非编码RNA。
在第三个方面中，提供一种抑制肿瘤细胞生长的方法，其包括使所述肿瘤细胞暴露于以KRM1为受体的肠道病毒，例如所述病毒选自CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12，具体地 是CV-A10病毒。
在一个实施方案中，所述肿瘤细胞例如是来自以下肿瘤的细胞：结肠癌，黑素瘤，前列腺癌，肺癌如非小细胞肺癌，肝癌、肝细胞癌、宫颈癌、子宫癌、胰腺癌、胃癌、食管癌、脑癌、甲母细胞癌和乳腺癌。
在另一个实施方案中，所述病毒是工程化的。例如工程化的病毒还包括或者能够产生有助于实现其目的的额外的蛋白质和/或其编码序列、和/或非编码核酸如非编码RNA，例如在这个方面中，术语“有助于实现其目的”是指有助于抑制肿瘤细胞生长。例如，所述工程化病毒的基因组还包括编码额外抗肿瘤剂或肿瘤治疗剂的核苷酸序列。
在第四个方面中，提供一种治疗肿瘤的方法，其包括向有此需要的对象施用以KRM1为受体的肠道病毒，更例如所述病毒选自CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12，例如是CV-A10病毒。
在一个实施方案中，所述肿瘤选自：结肠癌，黑素瘤，前列腺癌，肺癌如非小细胞肺癌，肝癌、肝细胞癌、宫颈癌、子宫癌、胰腺癌、胃癌、食管癌、脑癌、甲母细胞癌和乳腺癌。
在另一个实施方案中，所述病毒是工程化的。例如这种工程化可以是任何有助于实现其目的的工程化改造，例如在这个方面中，术语“有助于实现其目的”是指有助于治疗肿瘤。例如有助于病毒保存/运输，有助于施用病毒，有助于实现更好的抗病毒治疗效果。例如工程化的病毒还包括或者能够产生有助于实现其目的的额外的蛋白质、核酸和/或其编码序列，所述核酸例如是非编码核酸如非编码RNA。容易理解，本文术语“工程化”还可以包括为实现其他目的所实施的工程化改造。
在一个具体实施方案中，所述工程化病毒的基因组还包括编码额外肿瘤治疗剂的核苷酸序列。所述额外的肿瘤治疗剂，例如可以是治疗肿瘤的蛋白质和/或核酸如非编码RNA。
在另一个实施方案中，通过瘤内注射、癌旁注射、静脉注射和/或肌肉注射向所述对象施用所述病毒。例如通过瘤内注射施用所述病毒，通过癌旁注射施用所述病毒，通过静脉注射施用所述病毒，通过肌肉注射施用所述病毒，通过瘤内注射和/或癌旁注射施用所述病毒，通过瘤内注 射和/或静脉注射施用所述病毒，通过瘤内注射、癌旁注射和/或静脉注射施用所述病毒。瘤内注射是抗肿瘤病毒的典型施用途径，病毒可以不经循环系统由此直接进入治疗部位。但是瘤内注射经常需要有经验的专业人员来实施。癌旁注射则更容易操作，经初步培训的专业人员通常就有能力胜任。癌旁注射中，治疗剂一般比瘤内注射稍慢进入治疗部位，因此癌旁注射比瘤内注射也相对次选。不过在实际操作中，目标是瘤内注射的操作容易在实践中注射在癌旁。静脉注射是几乎所有医疗专业人员都掌握的操作，不需要额外培训。在静脉注射中，治疗剂随循环系统被带入治疗部位，进而治疗剂也将被循环系统稀释。因此，相对于瘤内注射和癌旁注射，静脉注射在抗肿瘤病毒治疗中一般是次选的。发明人意外发现，在使用本文的肠道病毒进行抗肿瘤治疗中，静脉注射几乎与瘤内注射有相同的抗肿瘤效果。肌肉注射对辅助条件要求是相对更低的，其需要经过局部吸收进入循环系统，然后再随循环系统进入待治疗部位。
在另一个实施方案中，通过消化道途径例如口服来施用所述病毒。众所周知，消化道途径不是抗肿瘤病毒治疗的通常选择。发明人意外发现，即便是通过消化道途径施用所述病毒，仍然可以实现显著的抗肿瘤效果。
在另一个实施方案中，所述方法还包括向所述对象施用额外的抗肿瘤治疗。所述额外的抗肿瘤治疗例如选自化学治疗、放射治疗、靶向治疗和免疫治疗中的一种或更多种。
需要指出的是，肠道病毒虽然是手足口病的病原体，但主要是CV-A16和EV-A71引起，而且只是在5岁以下儿童中较多见引起手足口病，多数患儿症状轻微，年长儿童和成年人很少见到有显著症状。基于本发明的意外发现，这种广泛的安全性使得以KRM1为受体的肠道病毒特别适用于治疗肿瘤，特别是较大儿童(大于5岁)和成年人中发现的肿瘤。因此，在一个具体实施方案中，所述治疗对象是较大儿童或成年人。
在第五个方面中，提供一种以KRM1为受体的肠道病毒，其用于抑制细胞中Wnt通路信号，用于使细胞中β-catenin降解，用于抑制肿瘤细胞如结肠癌细胞生长，或用于治疗肿瘤如结肠癌，例如所述病毒选自CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12，具体地 是CV-A10病毒。
在一个实施方案中，所述病毒是工程化的。例如这种工程化可以是任何有助于实现其目的的遗传工程改造，例如在这个方面中，术语“有助于实现其目的”是指有助于治疗肿瘤。例如有助于病毒保存/运输，有助于施用病毒，有助于实现更好的抗病毒治疗效果。例如工程化的病毒还包括或者能够产生有助于实现其目的的额外的蛋白质、核酸和/或其编码序列，所述核酸例如是非编码核酸如非编码RNA。容易理解，本文术语“工程化”还可以包括为实现其他目的所实施的遗传工程改造。
在一个具体实施方案中，所述工程化病毒的基因组还包括编码额外肿瘤治疗剂的核苷酸序列。所述额外的肿瘤治疗剂，例如可以是治疗肿瘤的蛋白质和/或核酸如非编码RNA。
在另一个实施方案中，通过瘤内注射、癌旁注射、静脉注射和/或肌肉注射向所述对象施用所述病毒。例如通过瘤内注射施用所述病毒，通过癌旁注射施用所述病毒，通过静脉注射施用所述病毒，通过肌肉注射施用所述病毒，通过瘤内注射和/或癌旁注射施用所述病毒，通过瘤内注射和/或静脉注射施用所述病毒，通过瘤内注射、癌旁注射和/或静脉注射施用所述病毒。瘤内注射是抗肿瘤病毒的典型施用途径，病毒可以不经循环系统由此直接进入治疗部位。但是瘤内注射经常需要有经验的专业人员来实施。癌旁注射则更容易操作，经初步培训的专业人员通常就有能力胜任。癌旁注射中，治疗剂一般比瘤内注射稍慢进入治疗部位，因此癌旁注射比瘤内注射也相对次选。不过在实际操作中，目标是瘤内注射的操作容易在实践中注射在癌旁。静脉注射是几乎所有医疗专业人员都掌握的操作，不需要额外培训。在静脉注射中，治疗剂随循环系统被带入治疗部位，进而治疗剂也将被循环系统稀释。因此，相对于瘤内注射和癌旁注射，静脉注射在抗肿瘤病毒治疗中一般是次选的。发明人意外发现，在使用本文的肠道病毒进行抗肿瘤治疗中，静脉注射几乎与瘤内注射有相同的抗肿瘤效果。肌肉注射对辅助条件要求是相对更低的，其需要经过局部吸收进入循环系统，然后再随循环系统进入待治疗部位。
在另一个实施方案中，通过消化道途径例如口服来施用所述病毒。众所周知，消化道途径不是抗肿瘤病毒治疗的通常选择。发明人意外发现，即便是通过消化道途径施用所述病毒，仍然可以实现显著的抗肿瘤 效果。
在第六个方面中，提供一种药物组合物，其包含第六个方面中所述的肠道病毒，用于抑制细胞中Wnt通路信号，用于使细胞中β-catenin降解，用于抑制肿瘤细胞如结肠癌细胞生长，或用于治疗肿瘤如结肠癌，例如所述病毒选自CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12，具体地是CV-A10病毒。
附图说明
图1示出了CV-A10的透射电镜图。标尺：100nm。
图2A示出了CV-A10体外抑制人结肠癌细胞RKO的生长，这种抑制作用与病毒滴度正相关，随时间延长而逐渐增强；图2B示出了EV-A71感染RKO细胞作为对照。
图3示出了在接种RKO结肠癌细胞的NCG小鼠中注射CV-A10病毒前后的肿瘤生长。(A)瘤内注射CV-A10病毒的统计学结果，(B)瘤内注射后肿瘤大小示意图，(C)静脉注射CV-A10病毒的统计学结果，(D)静脉注射后肿瘤大小示意图。
图4示出了重组KRM1蛋白的纯化和SDS-PAGE鉴定图。
图5示出了KRM1与CV-A10复合物的结构(A)，KRM1与CV-A10结合位点(B)，以及KRM1与DKK1结合位点(C)结构对比图。
图6示出了病毒感染细胞中报告基因的相对表达水平。CV-A10在RD细胞(A,B)和293T细胞(C,D)中对Wnt/β-catenin通路信号的下游基因表达起抑制作用，作为对照的EV-71则未表现出显著的抑制作用，DKK1表现出与CV-A10接近的抑制作用。
图7示出了CV-A10在RKO细胞中导致β-catenin的降解，这种作用与病毒量正相关，随病毒量的增加而增强。
图8示出了KRM1在癌症的不同阶段(A)及不同肿瘤(B)中的表达谱分析。
图9示出了CV-A10感染RKO细胞后KRM1表达增加。
图10示出了CV-A10抑制人肺癌细胞系MRC5、PC3、Calu-1和NCI-h1299的生长，这种抑制作用与病毒滴度正相关，随时间延长而逐渐增强。
具体实施方式
定义
除非特别指出，本文所用术语具有所属领域技术人员通常理解的含义。以下提供了本文中使用的一些术语的定义，如果对这些术语有其他解释，则以本文给出的定义为准。
本文所用术语“病毒”如“肠道病毒”包括直接从环境或患病个体分离的野生型病毒株，也包括从这些野生型病毒衍生的各种病毒变体。另一方面，除了常规感染产生的病毒颗粒，术语“病毒”还包括人为介入病毒复制、装配等一个或更多个过程产生的病毒颗粒和病毒样颗粒(VLP)。
本文所用术语“工程化的病毒”是指与野生型病毒不同的经过遗传工程改造的病毒或其变体，这种遗传工程改造可以是为了实现本文所述目的，也可以是为其他目的进行的遗传工程改造。
本文所用术语“治疗”是指有益于目标对象健康状况的任何措施，例如可以是预防疾病发生，降低疾病发生风险，减缓或阻碍疾病进程，抑制或停止疾病相关细胞或实体(如肿瘤细胞或肿瘤实体)的生长，杀死疾病相关细胞(如肿瘤细胞)，使疾病相关实体(如肿瘤实体)缩小，减小或消除疾病相关症状，防止或减缓疾病相关并发症的发生或发展，抑制或减缓疾病转移，提高患病对象生存率。
本文所用术语“抗肿瘤治疗”是指有助于预防或延迟肿瘤发生、降低肿瘤发生风险，减缓或阻碍肿瘤进程，抑制或停止肿瘤细胞或肿瘤实体的生长，杀死肿瘤细胞，使肿瘤实体停止生长或缩小，减小或消除肿瘤相关症状(如肿瘤疼痛)，防止或减缓肿瘤相关并发症的发生或发展，抑制或减缓肿瘤转移，提高患病对象生存率的任何措施。抗肿瘤治疗例如可以是手术切除、化学治疗、放射治疗、靶向治疗和免疫治疗中的一种或更多种。在医疗实践中，为了减缓或消除患病对象的不健康状态，医学专业人员经常选择一种或更多种适用的肿瘤治疗措施。
本文所用术语“Wnt通路”、“Wnt/β-catenin通路”是指经由Wnt及其受体蛋白介导的信号传递过程。Wnt通路是细胞内信号传递的一种重要通路，广泛参与了多种生理和病理过程。术语“抑制Wnt通路信号”是指使介导生理过程的Wnt通路信号发生与生理过程相反的变化，有时也称为负调节Wnt通路信号。
本文所用术语“选自”是指从多个候选项中选择。当适用时，在未特别指出的情况下，所述“选自”可以是从多个候选项中选择一种或更多种候选项。
实施例
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1：CV-A10病毒的扩增和纯化
柯萨奇病毒A10(CV-A10)HB09-035病毒株于2009年在河北省分离得到。该病毒株于2020年1月19日以保藏号CGMCC NO.19294保藏于中国普通微生物菌种保藏管理中心(中国北京市朝阳区北辰西路1号院3号)。人横纹肌肉瘤细胞(rhabdomyosarcoma cell，RD cell，ATCC CCL-136)培养在含10％胎牛血清的DMEM培养基中，5×10 7个细胞接种于15厘米培养皿中，37℃下5％CO 2培养24小时。弃去培养基，按感染复数MOI＝0.1加入CV-A10毒种，毒种用DMEM培养基稀释。继续培养36小时，当显微镜观察到90％细胞病变效应(CPE)时，收集上清液，使用
Figure PCTCN2021072775-appb-000001
TFF systems 300kD浓缩病毒，并去除细胞碎片。然后140,000g超速离心2小时，弃去上清。加入PBS(pH7.4)缓慢将病毒沉淀重悬，收集到微离心管中。之后将病毒重悬液通过15％-45％蔗糖密度梯度在140,000g离心4小时，每200μl为一层，分管收集，并加入等体积PBS降低溶液渗透压。将OD260/280大于1.5的样品利用透射电 镜进行检查，留取含成熟病毒颗粒较多的样品(如图1所示)。将病毒保存于PBS溶液中，测定浓度及滴度后备用。
实施例2：CV-A10抑制人结肠癌细胞系RKO生长
培养RKO结肠癌细胞系(ATCC CRL-2577)，取2×10 4个细胞置于96孔板中，培养8h。将培养液更换为无血清DMEM培养基，感染不同滴度的病毒(MOI＝0.001-10)，继续37℃培养，并在不同时间点测定细胞存活率。存活率测定使用CellTiter-Glo Luminescent Cell Viability Assay assays(Promega)试剂盒，按照使用手册进行检测。结果如图2A所示，感染高病毒滴度(MOI＝10)12h后开始出现细胞存活率出现下降的趋势，48h基本全部死亡。较低病毒滴度(MOI＝1)也于60h后致使癌细胞几乎全部死亡。其它滴度病毒在不同时间产生梯度的抑制效果。最低病毒滴度(MOI＝0.001)也在72h显示了对细胞生长的抑制现象。表明CV-A10可以在体外抑制结肠癌细胞系RKO生长，且与病毒滴度和感染时间正相关。用EV-A71在相同条件下进行实验，结果显示，EV-A71对人结肠癌细胞RKO直至48h仍无显著生长抑制作用(图2B)，这说明CV-A10的作用具有特异性。
实施例3：瘤内或静脉注射CV-A10抑制小鼠实体肿瘤生长
选用4周龄NCG小鼠(17-19g)共15只，背部注射5×10 6RKO细胞/只。种瘤后，饲养5天观察其背部成瘤情况。当肿瘤生长至120mm 3时，每3天治疗一次，即在第5，8，11，14，17天进行CV-A10病毒的瘤内注射或静脉注射，设置对照组，每组5只。其中：瘤内注射组为瘤内多点注射，静脉注射组为尾静脉单针注射，治疗剂量为1×10 7个病毒粒子/次。对照组使用PBS进行瘤内多点注射以排除操作误差。每隔两天记录小鼠的体重并用游标卡尺测量肿瘤的长径和短径，记录肿瘤的体积，连续监测20天。种瘤后20天处死小鼠，取出肿瘤，测量肿瘤的体积。从肿瘤体积数据可见：瘤内注射组在11天(经2次治疗，治疗后6天)、静脉注射组在第13天(经3次治疗，治疗后8天)时，肿瘤大小已出现显著差异。证明CV-A10瘤内注射(图3A-3B)或静脉注射(图3C-3D) 均可以抑制肿瘤生长。
实施例4：受体KRM1蛋白的表达和纯化
发明人将编码人Kremen 1(KRM1)胞外段(第23-373位氨基酸)的DNA序列(Genbank编号：AAH63787)通过限制性酶切位点HindIII和BamHI连接入pCMV3载体(北京义翘神州科技有限公司)。其中KRM1蛋白的5’端添加人II型干扰素信号肽，在3’端加入10个组氨酸的亲和标签(His10-tag)的编码序列并添加终止密码子(插入序列如SEQ ID NO.1所示)，获得质粒pCMV3-KRM1-ectodomain。接种培养1×10 7个293T细胞于含20ml DMEM培养基的15厘米细胞培养皿中，待细胞生长至70％，进行质粒转染。将50μg质粒和150μl转染试剂PEI(1mg/ml)配于缓冲液HBS(20mM HEPES，150mM NaCl，pH 7.4)中，转染体系共2ml，室温混合30min后加入培养293T细胞的15厘米培养皿中。转染6小时后弃去上清，将培养基更换为无血清无转染试剂的DMEM培养基中，继续培养3天。3天后收取培养上清，经镍离子亲和层析(HisTrap TM HP(GE))和凝胶过滤层析(superdex TM 200increase 10/300GL(GE))纯化后，通过SDS-PAGE鉴定蛋白纯度，获得可溶的人KRM1胞外段蛋白(如图4所示)。经鉴定，获得了高纯度KRM1蛋白，大小约为70kDa。
编码重组人KRM1胞外区的序列如下：
Figure PCTCN2021072775-appb-000002
Figure PCTCN2021072775-appb-000003
实施例5：KRM1与CV-A10结合模式与作用过程与DKK1相似
将实施例1和4中纯化的CV-A10和受体KRM1体外孵育，将已纯化的CV-A10病毒(2mg/ml)与过量的KRM1(0.2mg/ml)孵育5分钟，然后将复合物样品吸附在附有超薄碳膜的铜网上(Lacey carbon，中镜科仪)，吸附1分钟后，将铜网表面多余的病毒用滤纸吸干，借助Vitrobot Mark IV(FEI)冷冻制样机器迅速插入液态乙烷中，后转入液氮中保存样品。数据收集使用装有Gatan K2直接电子探测器的200kV Arctica(FEI)电镜完成。所有的电镜原始照片通过MotionCor2程序进行漂移校正。衬度传递函数(contrast transfer function，CTF)校正通过CTFFIND4计算，使用EMAN2进行颗粒自动挑选，最终通过Relion计算得到分辨率为
Figure PCTCN2021072775-appb-000004
Figure PCTCN2021072775-appb-000005
的CV-A10/KRM1复合物结构。最终分辨率通过傅里叶壳层关联函数(Fourier shell correlation，FSC)曲线估算得出。通过模拟生理条件细胞外pH 7.4的中性环境，确认KRM1通过其Kringle(KR)和WSC结构域与CV-A10病毒结合，其结合模式与KRM1在Wnt/β-catenin信号通路中 与DKK1结合的位点基本一致。如图5所示，通过对比KRM1与DKK1结合模式(PDB code：5FWW)表明CV-A10可以模拟DKK1抑制Wnt/β-catenin信号通路。
实施例6：CV-A10抑制Wnt/β-catenin通路信号转导
本实施例中，以实施例1中扩增纯化CV-A10类似的方法纯化了EV-A71病毒作为对照组。并参考实施例4类似的方法构建KRM1全长质粒，即将1-473位氨基酸编码序列(Genbank编号：AAH63787)通过5’XhoI和3’NotI限制性酶切位点连接入载体pLVX-DsRed-Monomer-N1(Clontech)中，并在3’端添加Flag标签和终止密码子，通过外源转染使细胞过表达KRM1。
将293T或RD细胞培养于24孔细胞培养板中，将报告基因质粒Top-Flash(Beyotime,D2501)、内参对照pRenilla-TK质粒转染进入细胞，其中一组同时加入构建的全长KRM1过表达质粒。转染体系为：每组3个重复孔，配制90μl HBS溶液，70μl PEI(0.1mg/ml)和2μg质粒。转染6小时后更换DMEM培养基。转染12小时后，向培养基中加入Wnt3a(R&D,1324-WN-500/CF)，并分别用CV-A10或EV-A71病毒(MOI＝1)感染细胞，处理24小时后裂解细胞进行荧光素酶活性检测。如图6所示，CV-A10对于Wnt/β-catenin信号通路有明显的抑制效果，且此作用在KRM1过表达时增强(图6B，6D)，基本阻断Wnt3a对于信号的激活作用。而EV-A71病毒对报告基因的表达无影响(图6A，6B)。因此CV-A10可以作为Wnt/β-catenin通路抑制剂发挥作用。此外，在293T组中加入DKK1蛋白(50ng/ml，购于北京义翘神州科技有限公司)，DKK1和CV-A10均表现出对Wnt/β-catenin信号通路的抑制作用，而两者同时加入时其抑制效果没有倍增(图6C，6D)。再次证实了CV-A10是通过病毒受体KRM1抑制Wnt/β-catenin信号通路，CV-A10与KRM1的结合表位与DKK1类似，二者存在竞争结合。
实施例7：CV-A10导致β-catenin的降解从而抑制Wnt/β-catenin通路
将RKO结肠癌细胞1×10 6培养于6孔板，12h后细胞长到90％时分 别感染CV-A10(MOI＝1)和不同滴度的病毒(MOI＝0.001-10)，继续37℃培养。感染CV-A10(MOI＝1)的RKO细胞在36h用PBS洗两次，加入IP裂解液裂解后进行Western blot检测。感染不同滴度病毒(MOI＝0.001-10)的细胞分别在12h、24h、36h、48h、60h、72h用PBS洗两次，用IP裂解液200μl裂解后进行Western blot检测。如图7所示，RKO细胞中β-catenin表达量随感染时间延长而降低(图7A)；在感染时间相同的情况下，β-catenin随加入病毒量的增加而降低(图7B)。这表明CV-A10抑制Wnt/β-catenin通路是通过β-catenin的降解来实现的。用CV-A10感染293T细胞获得了类似的结果(图7C)。
实施例8：CV-A10入侵关键受体KRM1的组织表达谱
利用The Cancer Genome Atlas(TCGA)数据库，分析发现KRM1在癌症感染不同时期表达水平无明显差异(图8A)。但值得注意的是，在不同肿瘤中KRM1表达量差异较大，在一些肿瘤例如肾嫌色细胞癌(KICH)、肺腺癌(LUAD)、肺鳞癌(LUSC)和肾透明细胞癌(KIRC)中具有较高表达，尤其在结肠癌中的表达量显著高于正常组织(图8B)。这提示CV-A10可能对这些肿瘤有更显著的治疗效果。
通过体外免疫荧光实验进一步发现，感染CV-A10后RKO细胞中KRM1表达量增加(图9)。这表明CV-A10感染细胞后引起细胞KRM1表达增加，会被更多的病毒感染，起正反馈作用。
实施例9：CV-A10降低肺癌细胞存活率
分别培养MRC5(ATCC,CCL-171)、PC3(ATCC,CRL-1435)、Calu-1(ATCC,HTB-54)、NCI-h1299(ATCC,CRL-5083)肺癌细胞系，2 10 4个细胞铺于96孔板中。培养8h后，换无血清DMEM培养基，感染不同滴度的病毒(MOI＝0.001-10)，继续37℃培养。使用CellTiter-Glo Luminescent Cell Viability Assay assays(Promega)试剂盒在不同时间点测定细胞存活率。如图10A-10D所示，4株肺癌细胞感染高病毒滴度(MOI＝10)12h后开始出现存活率下降的趋势，48h基本全部死亡。较高病 毒滴度(MOI＝1)于60h后致使癌细胞几乎全部死亡。其它滴度病毒在不同时间产生梯度的抑制效果。最低病毒滴度(MOI＝0.001)也在72h出现了对细胞生长的抑制现象。表明CV-A10可以导致肺癌细胞的存活率下降，且其效果与病毒滴度和感染时间正相关。

Claims (14)

  1. 一种抑制细胞中Wnt通路信号的方法，其包括使所述细胞暴露于以KRM1为受体的肠道病毒，例如所述病毒选自CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12，具体地是CV-A10。
  2. 一种使细胞中β-catenin降解的方法，其包括使所述细胞暴露于以KRM1为受体的肠道病毒，例如所述病毒选自CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12，具体地是CV-A10。
  3. 一种抑制肿瘤细胞如结肠癌细胞、肺癌细胞生长的方法，其包括使所述肿瘤细胞暴露于以KRM1为受体的肠道病毒，例如所述病毒选自CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12，具体地是CV-A10。
  4. 一种治疗肿瘤如结肠癌、肺癌的方法，其包括向有此需要的对象施用以KRM1为受体的肠道病毒，例如所述病毒选自CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12，具体地是CV-A10。
  5. 根据权利要求1至4任一项的方法，其中所述病毒是工程化的。
  6. 根据权利要求5的方法，其中所述工程化病毒的基因组还包括编码额外肿瘤治疗剂的核苷酸序列。
  7. 根据权利要求4至6任一项的方法，其中通过消化道途径和/或注射途径向所述对象施用所述病毒，例如通过选自口服、瘤内注射、癌旁注射、肌肉注射和静脉注射的途径施用所述病毒。
  8. 根据权利要求4至7任一项的方法，其还包括向所述对象施用额外的抗肿瘤治疗。
  9. 根据权利要求8的方法，其中所述额外的抗肿瘤治疗选自化学治疗、放射治疗、靶向治疗和免疫治疗。
  10. 一种以KRM1为受体的肠道病毒，其用于抑制细胞中Wnt通路信号，用于使细胞中β-catenin降解，用于抑制肿瘤细胞如结肠癌细胞、肺癌细胞生长，或用于治疗肿瘤如结肠癌、肺癌，例如所述病毒选自CV-A2，CV-A3，CV-A4，CV-A5，CV-A6，CV-A10和CV-A12，更具体地是CV-A10。
  11. 根据权利要求10的病毒，其是工程化的。
  12. 根据权利要求10或11的病毒，其基因组还包含编码额外肿瘤治疗剂的核苷酸序列。
  13. 根据权利要求10的病毒，其于2020年1月19日以保藏号CGMCC NO.19294保藏于中国普通微生物菌种保藏管理中心。
  14. 一种药物组合物，其包含权利要求10至13任一项所述的病毒，优选地所述药物组合物用于治疗肿瘤如结肠癌或肺癌。
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