TECHNICAL FIELD

The present disclosure belongs to the technical field of biomedicine, and it relates to use of alphavirus in preparation of antitumor drugs.

BACKGROUND OF THE DISCLOSURE

Tumors derive from accumulative change of genes and epigenetics in normal cells, such change promotes conversion of normal cells into malignant tumors. This complex pathological change process determines diversity of mechanisms of genesis, maintenance and metastasis in different tumors. At present, surgical excision, chemotherapy and radiotherapy are common methods for clinical therapy of tumors, however surgical excision of tumors is prone to recur, and toxic and side effects of the radiotherapy and the chemotherapy are obvious.

15-20% of human cancers are associated with viral infection, for example, hepatitis B virus (HBV) and hepatitis C virus (HCV) are associated with liver cancers, and human papillomavirus (HPV) is associated with cervical cancers, etc.

Alphavirus belongs to Togaviridae, and it is a type of single stranded RNA virus with an envelope structure, transmitted mainly by a transmission media of arthropod. 13 in 29 types of alphaviruses can cause diseases of human and animals (David M. Knipe, Peter M. Howley, Chapter 23, Alphaviruses, Fields Virology 6thedition: 651-685, 2013).

Venezuelan equine encephalitis virus of alphaviruses can act as a vector to transduce dendritic cells so as to treat tumors (Moran T P, Burgents J E, Long B, et al: Alphaviral vector-transduced dendritic cells are successful therapeutic vaccines against neu-overexpressing tumors in wild-type mice. Vaccine 25: 6604-6612, 2007). However, such virus had induced fever, convulsion, abortion and even death of human, thus selectivity and safety problems severely affect use of the virus in antitumor therapy.

SUMMARY OF THE DISCLOSURE

A purpose of the present disclosure is to provide a safe and effective viral antitumor drug.

A further purpose of the present disclosure is to provide a safe and effective viral antitumor drug against a specific tumor type.

A further purpose of the present disclosure is to provide a safe and effective viral antitumor drug against a specific individual/tumor.

A further purpose of the present disclosure is to provide an effective antitumor administrating system and administrating method.

A further purpose of the present disclosure is to provide a more effective antitumor drug and a tumor treatment method.

The above-mentioned purposes are achieved by the present disclosure via the following technical solution.

The present disclosure provides use of alphavirus in preparation of antitumor drugs, wherein the alphavirus is M1 virus or Getah virus.

M1 virus (Alphavirus M1) belongs to Alphavirus, and it was isolated fromCulexmosquitos in Hainan Island, China in 1964 (Li X D, et al: Isolation of Getah virus from mosquitos collected on Hainan Island, China, and results of a serosurvey. Southeast Asian J Trop Med Public Health 23: 730-734, 1992.). Complete genomic sequence of M1 virus was determined in 2008 (Zhai Y G, et al: Complete sequence characterization of isolates of Getah virus (genus Alphavirus, family Togaviridae) from China. J Gen Virol 89: 1446-1456, 2008.). Its acquisition method is optional but not limited to the acquisition by the method in the above-mentioned literatures, or by the following deposit information (deposit number: CCTCC V201423; deposit time: Jul. 17, 2014; Classification and nomenclature: Alphavirus M1; depositary institution: China Center for Type Culture Collection; deposit address: Wu Han University in Luo Jia Shan, Wuchang District, Wuhan, Hubei Province).

The results of the previous researches on M1 virus by researchers of the present disclosure indicated that M1 virus had a killing effect against some tumor cells, such as rat malignant glioma cell C6, human malignant glioma cell U251 and U-87; however, it was not effective in killing some other tumor cells, such as human malignant glioma cell T98G. These researches cannot confirm that M1 virus has an effective antitumor effect.

The present disclosure further provides tumor types to which the virus is more applicable, in order to improve the therapy efficacy when M1 virus is used as the antitumor drug.

More preferably, the present disclosure using M1 virus as the antitumor drugs is effective in treating one or more of liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, and gastric cancer.

The inventors found that M1 virus causes varying degrees of cell death for various tumor cells. After 48 hours of M1 virus treatment of the tumor cells (MOI=10), the cell death rates of pancreatic cancer, nasopharyngeal carcinoma, prostate cancer and melanoma are more than 50%; the cell death rates of colorectal cancer (LoVo, HCT-8, SW620 and SW480), liver cancer (Hep3B, Huh-7 and Huh-6), bladder cancer and breast cancer are more than 40%; the cell death rates of glioma, cervical cancer, lung cancer are more than 30%; the cell death rate of gastric cancer is more than 20%. The above-mentioned results suggest that the M1 virus as an antitumor drug has the most significant effects on such tumor types: pancreatic cancer, nasopharyngeal carcinoma, prostate cancer and melanoma; while following are tumor types of colorectal cancer, liver cancer, bladder cancer and breast cancer; while further following are tumor types of glioma, cervical cancer, lung cancer; while least significant effects occur with the tumor type of gastric cancer.

Because M1 virus belongs to Getah-like virus and its homology with Getah virus is up to 97.8%, a person skilled in the art have a reason to recognize that, on basis of antitumor effect of M1 virus, Getah virus also has a similar action and effect as M1 virus.

Further, the present disclosure provides a method for more accurately and effectively providing a therapeutic scheme and a therapeutic drug against a specific individual/tumor, as well as an associated drug against the specific individual/tumor.

The inventors first found that the virus was suitable for treating ZAP low expression tumors or ZAP negative tumors, preferably for treating ZAP low expression solid tumor or ZAP negative solid tumor.

It found that the efficacy of M1 virus therapy against tumor is closely related to the ZAP expression regulation of the tumor. The replication of M1 virus is inhibited by ZAP which is low expressed or negative in various tumor types. M1 virus can selectively treat ZAP low expression or ZAP negative tumors/individual.

ZAP is an abbreviation of zinc Finger CCCH-type antiviral protein 1, its English name is zinc finger CCCH-type antiviral protein 1, and it is encoded by zc3hav1 gene. It is reported that, in cells ZAP inhibits replication of certain viruses, such as Ebola virus and Marburg virus, by mechanism of inducing RNA degradation and translation inhibition. However, ZAP has no inhibitory effect on replication of other viruses, such as vesicular stomatitis virus, poliovirus and yellow fever virus, etc.

The inventors found that M1 virus can significantly cause cell death in ZAP low expression/ZAP negative cell lines, and M1 virus was enriched in ZAP low expression/ZAP negative tumor tissues of a tumor-bearing animal body, inhibiting tumor growth. In the meanwhile M1 virus inhibits survival of ZAP low expression/ZAP negative human ex vivo living tumor tissues.

The inventors found for the first time that in different multiple types of tumors, expression level of ZAP in tumor tissues is lower than that in paracancerous non-neoplastic tissues. Immunohistochemical analysis on various types of clinical tumor pathological specimens indicated that, in 69% of liver cancer tissues, 52% of the colorectal cancer tissues and 61% of the bladder cancer tissues, ZAP expression levels are significantly lower than that in the corresponding paracancerous non-neoplastic tissues. M1 virus can be used for selectively treating ZAP low expression/ZAP negative tumors.

The experiment results proved for the first time that replication of M1 virus was inhibited by ZAP, and ZAP expression level was the decisive factor influencing the M1 virus' effect of selective tumor cell death induction and tumor growth inhibition. The inventors found that antitumor effect of M1 virus was directly related to the expression level of ZAP. The inventors found that, if the tumor cells with knockdown of ZAP were infected with M1 virus, the survival rate of the tumor cells was significantly decreased compared with the tumor cells without knockdown of ZAP. Thus, as an optional preferred therapeutic scheme, when a cancer patient is treated with M1 viral, a ZAP inhibitor can be administrated at the same time or in advance, in order to improve sensitivity of tumor to M1 virus.

Therefore, in order to further improve therapy efficacy of M1 virus as an antitumor drug, when adopting a therapeutic scheme, the ZAP expression in tumor can be firstly determined for the patient, then the therapeutic scheme using M1 virus can be specifically administrated, thereby improving the efficacy of the therapeutic scheme, and avoiding time delay due to ineffective administration and avoiding drug abuse. For example, the tumor ZAP expression of the patient is firstly detected before administration, and if the tumor is of ZAP low expression or ZAP negative expression, the M1 viral therapy can be directly provided; if the tumor is of ZAP normal expression/ZAP high expression, a ZAP inhibitor can be provided before or at the same time during the administration of the M1 virus, thereby improve sensitivity of the tumor to M1 virus, and improve the therapy efficacy. Said ZAP inhibitor is for example, ZAP expression or function inhibitor, ZAP interference fragment, or ZAP antibody, etc. ZAP expression quantity in the tumor directly affects the efficacy of the M1 therapy. The lower ZAP expression quantity of the tumor is, the more effective it is to be treated by M1. To determine whether a certain individual/tumor is suitable for M1 treatment or not, tumor ZAP expression level can firstly be detected. Preferably, ZAP expression level can be determined by, but not limited to the following means:

Low or high expression of ZAP refers to conclusion obtained by comparison of ZAP mRNA or protein quantity between two groups of specimens or two specimens. If the ZAP mRNA or protein quantity of one group of specimens or one specimen is less than or more than that of the other group of specimens or the other specimen, this specimen is called ZAP low or high expression, respectively; ZAP negative refers those totally with no expression of ZAP mRNA or protein. Sets of specimens used for comparison of ZAP mRNA and protein quantity can be: tumor cells vs. normal cells, tumor tissues vs. paracancerous non-neoplastic tissues, or tumors effective to M1 treatment vs. tumors that are not effective to M1 treatment.

As an optional means, ZAP high, low or negative expression in the tumors means that, the quantity of ZAP mRNA and protein of the tumor tissues is more, less or no expression, respectively, compared with that of the corresponding paracancerous non-neoplastic tissues. If the normalized expression quantity of ZAP mRNA or protein of the former one (tumor tissues) is less than that of the latter one (paracancerous non-neoplastic tissues), in other word, if the ratio of the ZAP normalized expression quantity of the tumor tissues to that of the paracancerous non-neoplastic tissues is <1, it belongs to ZAP low expression, and thus is suitable to be treated by M1. More effectively, the therapy object is a tumor in which the ratio of ZAP normalized expression quantity of the tumor tissues to that of the paracancerous non-neoplastic tissues is <0.8, more preferably <0.6, more preferably <0.4, more preferably <0.3, more preferably <0.2, more preferably <0.1, and most preferably, the tumor tissue is ZAP negative. These tumor tissues and the corresponding paracancerous non-neoplastic tissues include, but not limited to, tissue specimens obtained from pathological puncture or surgical resection. Clinical investigation found that, in certain tumor tissues, ZAP expression quantity is even higher than that in the paracancerous non-neoplastic tissue, and these tumors or tumor patients will not be suitable for a direct administration of M1 for therapy. The detection method for ZAP mRNA or protein includes but not limited to QRT-PCR, Northern Blot, Western Blot, immunohistochemistry, ELISA, etc. To accurately determine the difference of ZAP mRNA or protein quantity between different specimens, the normalized expression quantity of ZAP mRNA or protein in each specimen are firstly calculated. The normalized expression quantity refers to that ZAP mRNA or protein expression value of each specimen is divided by mRNA or protein expression value of a internal reference of the specimen, and a normalized analysis is conducted to obtain a ZAP normalized expression quantity of the specimen. In different detection methods, the internal references can be different, and their common characteristics is that the internal reference expression quantities in different cells or tissue specimens are identical, therefore ZAP expression quantities of different specimens through such normalized analysis are comparable, so as to determine the quantity difference of ZAP mRNA or protein between the specimens.

In one exemplary example of the present disclosure (FIG. 4), for the growth inhibition effects of M1 against human ex vivo cultivated living liver cancer tissues and colorectal cancer tissues, different inhibition results are obtained between the two kinds of tissues (Table 2 and Table 3), wherein in total up to 32 specimens present a tumor growth inhibition rate of more than 10%, while 19 specimens present a tumor growth inhibition rate of less than or equal to 10%. The expression levels of ZAP and internal reference mRNA of each tumor tissue in these two groups are further analyzed by QRT-PCR method (respectively 2−Ct value), and 2−Ct-ZAPof each specimen is divided by 2−Ct-internal referenceto obtain respective ZAP normalized expression quantity. Based on a statistical analysis of ZAP normalized expression quantity of the specimens in the above-mentioned two groups, it is found that the ZAP normalized expression quantity of the specimen group with an inhibition rate of more than 10% is 0.117±0.890, it is lower than the group with an inhibition rate of less than or equal to 10% (0.791±0.108), and the ratio of these two mean value is 0.148.

As another optional method, ZAP high, low or negative expression of the tumor means that the quantity of ZAP mRNA and protein of the tumor cells (e.g., derived from tumor patient's cultured tumor cells) is more, less or no expressed, compared with those of normal cells. If the ZAP mRNA or protein normalized expression quantity of the former is less than that of the latter (i.e., the ratio of ZAP normalized expression quantity of the tumor cells to that of the normal cells is <1), it belongs to ZAP low expression, and it is suitable for a therapy with M1. A more effective therapy object is a tumor in which the ratio of ZAP normalized expression quantity of the tumor cells to that of the normal cells is <0.8, more preferably <0.6, more preferably <0.4, more preferably <0.3, more preferably <0.2, more preferably <0.1, and most preferably, the tumor cell is ZAP negative.

In an exemplary example of the present disclosure (FIG. 3c), difference of ZAP protein expression level between HepG2 liver cancer cell line and L-02 normal liver cell line is detected by Western Blot method, meanwhile a standard reference β-actin whose expression quantity is identical between different specimens is detected, wherein grey scale of Western blot detection band represents the quantity of the detected molecules. The ZAP normalized protein expression quantity=(ZAP band grey scale mean value)/(β-actin band grey scale mean value). The ratio of ZAP normalized protein expression quantity of HepG2 to those of L-02 is 0.8, ZAP is low expressed in Hep G2. After infection with the M1 virus, the survival rate of the Hep G2 cell is only 70.4%±3.5%, while the survival rate of L-02 under same treatment is 100.3±10.0%, the difference of survival rate between them is of statistical significance.

In another exemplary example (FIG. 3aandFIG. 3b), for ZAP of tumor cell lines T24, SCaBER, LoVo and Hep3B, the mRNA (FIG. 3a) and protein (FIG. 3b) are detected by QRT-PCR and Western blot, and then compared with respective internal references, to obtain the normalized expression quantity which is undetectable or close to 0 (<0.1), that is to say, the ZAP expression is negative or close to negative (<0.1); after these tumor cells are infected by M1, cell death is induced, and the cell survival rate is significantly reduced to: T24 21.1%, SCaBER 11.5%, LoVo 6.9% and Hep3B 3.8% (Table 1). For the normal cell L-02 and HEB as shown inFIG. 3aandFIG. 3b, the ratio of the normalized expression quantity of ZAP mRNA (FIG. 3a) or protein to that of the above-mentioned tumor cell is more than 1, which belongs to ZAP high expression, and after M1 infection no obvious reduction of survival rate of these normal cells is induced. The survival rate of L-02 is 100.3%, and that of HEB is 98.8% (Table 1).

Thereby, the present disclosure also provides an antitumor administrating system, characterized in that, it comprises a ZAP expression level detecting reagent, and alphavirus; the alphavirus is M1 virus or Getah virus. The tumor ZAP expression level of the patient is firstly detected, then a suitable administration scheme is specifically adopted.

The present disclosure also provides an antitumor drug, comprising alphavirus and ZAP inhibitor; the alphavirus is M1 virus or Getah virus. The ZAP inhibitor is ZAP expression or function inhibitor, ZAP interference fragment, or ZAP antibody, etc.

In order to avoid killing effect of M1 virus to normal cells, preferably, the ZAP inhibitor is specifically provided or targeted to the tumor tissues, being a tumor targeted ZAP inhibitor.

As an optional embodiment, antitumor drug provided by the present disclosure can be injectio, tablet, capsule, or patch, etc. As a preferred embodiment, the antitumor drug of the present disclosure is injectio; preferably, intravenous injection is conducted.

As an optional administration means, the M1 virus of the present disclosure can be administered by intravenous or intratumoral injection. In the intratumoral injection, 2×105PFU/kg-2×109PFU/kg is administrated every day; in the intravenous injection, 2×106PFU/kg-2×1010PFU/kg is administrated every day. Compared with the solvent control group, the M1 virus group significantly inhibited the growth of tumors.

Compared with the prior art, the present disclosure has the following beneficial effects:

The antitumor drugs provided by the present disclosure may be used for treating various types of tumors including liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, and gastric cancer. Cytology experiments prove that M1 virus causes death of various types of tumor cells; animal experiments prove that M1 virus in vivo significantly inhibits growth of liver cancer and colorectal cancer; human ex vivo living tumor tissue culture experiments prove that M1 virus significantly inhibits survival of liver cancer and colorectal cancer tissues.

The antitumor drugs provided by the present disclosure can preferentially treat ZAP low expression/ZAP negative tumors, including but not limited to liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, and gastric cancer.

The antitumor drug provided by the present disclosure has selective antitumor activity, with good safety. M1 virus can selectively cause tumor cell death, but it has no effect on survival of the normal cells, indicating that M1 virus has tumor cell selectivity. In tumor-bearing nude mouse body, the M1 virus intravenously injected is highly enriched in the tumor tissues, while the virus quantity existed in the normal tissues is lower, and the difference of the virus quantity between the above said two kinds of tissues is about 102-106times. This further proves that M1 virus selectively affect tumor. In addition, the administration of the M1 virus does not affect body weight and mental status of the nude mouse, indicating that the M1 virus is with good safety.

The present disclosure for the first time provides a safe and effective viral antitumor drug against specific individual/tumor. The drug of the present disclosure selectively treats ZAP low expression/ZAP negative tumors, thereby increasing dosage effective rate, avoiding ineffective administration and drug abuse.

The present disclosure provides a more effective administrating method and administrating system. The ZAP expression level of the patient tumor is detected firstly, then a drug therapy is specifically provided, or assisted by other means to provide a therapy, thereby improving specificity and efficacy of the M1 virus therapy.

The present disclosure provides a more effective antitumor drug and tumor therapeutic method; a ZAP inhibitor is supplemented before administration or at the same time of administration, thereby improving sensitivity of the tumors to the drug.

