Patent Description:
Malignant tumors are major diseases that threat human health and survival in today's society. According to the statistical data of World Health Organization (WHO), there are about <NUM> million new patients with cancers in the world in <NUM>, and about <NUM> million patients died of cancers. In China, according to the data released by China's Cancer Registration Center in <NUM>, there are about <NUM> million new cases of cancers annually, and about <NUM> million of cases die. Tumor metastasis is the most important cause of death for patients with cancers. Tumor metastasis is a complex, multi-step biological process, in which cancer cells evolve to metastasize through the circulatory system and survive in a distant site, forming the tumor of the same type as the primary tumor. Metastasis, as a major malignant manifestation of the tumor, seriously affects the treatment efficacy and prognosis of cancer patients. Approximately <NUM>% of patients have metastasis when primary tumor is diagnosed clinically. The tumor metastasis is the biggest killer of cancer patients, and also the biggest bottleneck in cancer treatment. Among the wide range of anti-cancer drugs in today's market, most of the clinically used first-line chemotherapy drugs (such as doxorubicin, doxorubicin, paclitaxel, etc.) affect the survival, proliferation, vascular survival of tumors, but rarely target tumor metastasis. Moreover, the only few anti-metastatic drugs that are available have limited efficacy due to great side effects and drug resistance, etc.. Therefore, there is a long-term urgent need of anti-metastatic drugs on the market, and it is of great significance to develop the drugs to treat tumor metastasis.

EZH2 (the enhancer of zeste homologue <NUM>) is the functional enzymatic component of the polycomb repressive complex <NUM> (PRC2), which is involved in chromatin condensation by adding three methyl groups to histone H3 at lysine <NUM> to inhibit the transcription of relevant gene (e.g. tumor suppressor gene). Studies have shown that EZH2 and histone H3K27 methylation are closely related to cancers. The high expression of EZH2 was found in lymphoma, metastatic prostate cancer and breast cancer, and was associated with breast cancer invasion. In addition, EZH2 is overexpressed in many human malignancies, such as lung cancer, lymphoma, leukemia, pancreatic cancer, cervical cancer, colon cancer, liver cancer, gastric cancer, melanoma, kidney cancer, bladder cancer, etc., and its expression level is significantly increased in metastatic tumors, often positively correlated with poor prognosis. Preclinical researches showed that the drugs targeting EZH2 can inhibit the progression of brain cancer and prostate cancer. Thus, EZH2 could serve as a potential drug target for metastatic tumor therapy: the treatment of tumors is achieved by downregulating the expression and activity of EZH2, reducing the histone methylation and enhancing the expressions of tumor suppressor genes.

With the development of the genetic engineering and targeting technology of bacteria and virus, there are more and more studies on the bacterial treatment of cancers since the middle of <NUM>. Researchers have found that typhoid salmonella can be used as a good gene vector to effectively kill tumor cells in the body of mice in a targeted manner. Salmonella is a group of gram-negative, invasive intracellular facultative anaerobes that are parasitic in intestines of humans and animals. VNP20009 is an attenuated Salmonella typhimurium strain with deletion of msb B and pur I genes. It is genetically stable and susceptible to antibiotics. The msb B gene is necessary for the lipid acylation to form endotoxin, and its deletion prevents the lipid A terminal from being acylated and reduces toxicity. The pur I gene is involved in purine metabolism. Bacteria of pur I deletion needs exogenous adenine to reproduce. VNP20009 also reduces tumor necrosis factor (TNF) induced by typhoid salmonella, resulting in reduced inflammatory response. Therefore, the low pathogenicity of VNP20009 enhances its safety for clinical treatment. VNP20009 has been widely used in cancer studies. It can act on a variety of mouse solid tumor models, including melanoma, lung cancer, colon cancer, breast cancer, kidney cancer. One of the major advantages of VNP20009 as a tumor gene therapy vector is that it can aggregate at the tumor sites in a highly targeted manner. Researchers have found in a variety of mouse models of solid tumors that the amount of VNP20009 in the tumors is higher than that in the major organs such as liver by <NUM>~<NUM> times. VNP20009 can aggregate and reproduce in priority in the hypoxic necrosis zone of tumor tissues. And within the same period of time, the passage number of bacteria in the tumor tissues is significantly higher than that in normal tissues, making attenuated Salmonella as a new anti-tumor agent and a vector of tumor targeted therapy. The possible mechanism of slowed tumor growth caused by Salmonella: the nutrients required for tumor growth are consumed by the bacteria, and the enzymes produced by the bacteria such as asparaginase, can deplete the essential amino acids required for tumor growth; the local toxins or tumor necrosis factor α secreted by the bacteria to the extracellular microenvironment can affect tumor angiogenesis; in addition, non-specific inflammatory responses at the site of bacterial growth can potentially activate anti-tumor T cells. However, the inhibitory effect of VNP20009 on tumor metastasis has not been found yet.

Tumor cells require adequate nutrition in order to maintain its high rate of reproduction. In addition to carbohydrates, the need for methionine (Met), glutamine, and arginine is particularly high. Previous studies have established that Met-dependency is a common feature of most tumor cells, such as breast cancer, lung cancer, colon cancer, kidney cancer, bladder cancer, melanoma, glioma, etc. High Met-dependency does not exist in normal cells. Both in vivo and in vitro experiments have confirmed that dietary intervention with methionine deficiency can delay the proliferation of tumor cells. However, long-term deficiency of Met can cause malnutrition, metabolic disorders, and aggravate tumor growth due to a long-term DNA hypomethylation. Thus, by specifically degrading Met to methylselenol, α-ketobutyrate and ammonia through L-methioninase and lowering the level of methionine in vivo, we will be able to effectively inhibit the growth of tumor cells or even degrade them. However, since methioninase is not expressed in mammal itself, the exogenous administration may have some side effects, often causing the body's immune response. Methionine is an essential amino acid, which produces S-adenosylmethionine (SAM) under the catalysis of methionine adenosylytransferase. SAM, also known as active methionine, is the most important methyl donor in vivo, which is involved in the methyl transfer catalytic reaction of various substances such as DNAs, proteins in the body. EZH2 can transfer the active methyl group of SAM to specific amino acids of histone, so as to involve in the epigenetic modification of chromosomes, and inhibit the transcription of related genes. Previously we have prepared attenuated Salmonella strain VNP20009-M that expresses methioninase which can promote the apoptosis of breast cancer, pancreatic cancer, prostate cancer, with good anti-tumor effect. We have submitted patent applications (patent number <CIT>, <CIT>,<CIT>), of which, the patent application <CIT> has been granted right of patent. The methioninase can decompose the content of methionine in vivo, which can not only promote tumor cell apoptosis, but may also be effective in inhibiting the EZH2 activity through reducing the SAM content, thereby inhibiting tumor metastasis. Zhou et al. , validly found hypotoxicity and tumor-targeting ability of attenuated genetically modified Salmonella typhymurium as a vector for cancer gene therapy with great potential as a vector for cancer gene therapy (<NPL>). <CIT> discloses an attenuated Salmonella typhimurium genetically engineered bacterium for preparation of treatment of prostatic cancer. The genetically engineered bacterium has tumor targeting property and an obvious suppression effect on prostate cancer cells. The genetically engineered bacterium with plasmids cloned with an L-methioninase gene can continuously express L-methioninase in a tumor tissue. <CIT> discloses a genetically engineered bacterium for treatment of breast cancer, acquired by subcloning methionine enzyme genes in pSVSPORT particles, then by using the particles to transform attenuated Salmonella typhimurium VNP20009. <CIT> inter alia discloses the isolation and use of tumor-specific, suicide gene-containing strains of Salmonella typhimurium in treatment of solid tumors.

The technical problem of the invention is to provide uses of genetically engineered strain VNP20009-M in manufacturing biological medicines for preventing and treating cancer metastasis.

To solve the above technical problems, the invention adopts the following technical solutions.

A genetically engineered strain VNP20009-M for use in the prevention and treatment of cancer metastasis in a human subject, wherein the VNP20009-M is an attenuated Salmonella typhimurium genetically engineered strain VNP20009 cloned with an L-methioninase gene. The cancer is prostate cancer, pancreatic cancer, breast cancer, liver cancer or leukemia.

In certain embodiments, the genetically engineered strain comprises a plasmid cloned with an L-methioninase gene.

In certain embodiments, the genetically engineered strain VNP20009-M is constructed according to the following method: subclone the L-methioninase gene into a plasmid to obtain L-methioninase expression plasmid, then electro-transform the L-methioninase expression plasmid to attenuated Salmonella typhimurium VNP20009, to get the VNP20009-M.

In certain embodiments, the plasmid includes, but not limited to, a pSVSPORT plasmid, a pTrc99A plasmid, a pcDNA3. <NUM> plasmid, a pBR322 plasmid or a pET23a plasmid.

Most preferably, in the process of constructing genetically engineered strain VNP20009-M, the L-methioninase gene is subcloned into the plasmid by Kpn I and Hind III restriction sites when the pSVSPORT plasmid is used, to get the L-methioninase expression plasmid, and then the L-methioninase expression plasmid is electro-transformed into attenuated Salmonella typhimurium VNP20009, to get the genetically engineered bacterium.

In certain embodiments, the electrotransformation condition is as follows: voltage 2400V, resistance <NUM>Ω, capacitance <NUM>µF, discharge time <NUM>.

The routes of administration for the prevention and treatment of cancers include, but not limited to, oral administration, topical administration, injection administration (including but not limited to intravenous, peritoneal, subcutaneous, intramuscular, intratumoral administration), etc..

The engineered strain VNP20009-M of attenuated Salmonella typhimurium in capable of inhibiting Histone methyltransferase (HMT) EZH2 in a cell.

In certain embodiments, the L-methioninase and genetically engineered strain VNP20009-M are constructed according to the following method: subclone the L-methioninase gene into a plasmid to obtain L-methioninase expression plasmid, then transform the L-methioninase expression plasmid into E. coli and purify to get the methioninease protein, or electro-transform the L-methioninase expression plasmid to attenuated Salmonella typhimurium VNP20009, to get the VNP20009-M.

The HMT EZH2 inhibitor can be administered via a verity of routes, including but not limited to oral, topical, injection administration (including but not limited to intravenous, peritoneal, subcutaneous, intramuscular, intratumoral administration), etc..

Compared with prior art, the invention has the following advantages:.

The present invention can be better understood from the following examples. However, it will be readily understood by those skilled in the art that the embodiments described are intended to be illustrative of the invention, not and should not be construed as limiting the invention as set forth in the claims.

The L-methioninase (GenBank: L43133. <NUM>) gene was synthesized and subcloned to pUC57 plasmid (Genscript), then subcloned to pSVSPORT plasmid (invitrogen) through the Kpn I and Hind III restriction sites, to get the pSVSPORT-L-methioninase expression plasmid. The specific procedure is as follows:
The pSVSPORT plasmid was digested with Kpn I and Hind III, with the digestion system: 2µg of plasmid DNA, <NUM> of <NUM>× buffer, <NUM>. 5µL of Kpn I enzyme, <NUM>. 5µL of Hind III enzyme, added ddH<NUM>O to 30µL, incubate for <NUM> at <NUM>, then the digests was separated by <NUM>% agarose gel electrophoresis, to cut out DNA bands with the size of <NUM> kb, then DNA was purified by gel recovery and purification kit.

DNA fragments of L-methioninase coding region were obtained by gene synthesis and subcloned to pUC57 plasmid (Genscript), digested with Kpn I and Hind III, with the digestion system: 3µg of plasmid DNA, <NUM> of <NUM>× buffer, <NUM>. 5µL of Kpn I enzyme, <NUM>. 5µL of Hind III enzyme, added ddH<NUM>O to 30µL, warm bath for <NUM> at <NUM>, then the digestion system was separated by <NUM>% agarose gel electrophoresis, to cut out DNA bands with the size of <NUM> kb, then DNA was purified by gel recovery and purification kit.

The pSVSPORT (Kpn I/Hind III) and DNA fragment of the L-methioninase coding region (Kpn I/Hind III) were ligated. The ligation reaction condition: <NUM> of vector, 6µL of inserted fragment, 1µL of T4 DNA ligase, water bath for <NUM> at <NUM>.

The ligation product was transformed into competent cells of E. coli DH5a (Takara). One tube of <NUM> DH5a competent cells was placed on the ice until thawing, then <NUM>µL of above ligation product was added, mixed well by flicking, incubated on ice for <NUM>; after heat shock <NUM> at <NUM>, placed on ice for <NUM>; then <NUM>µL of non-resistant LB liquid medium was added and incubated at <NUM> for <NUM> with shaking, then spread on ampicillin resistant LB medium plate and cultured overnight.

When clones grew, single clone was innoculated to <NUM> of ampicillin-containing LB medium, incubated at <NUM> for <NUM>. The plasmid DNA was extracted and identified by Kpn I and Hind III digestion. Two DNA bands at the size of <NUM> kb, <NUM> kb were obtained in the positive clones, as shown in <FIG>. The sequence of the positive clones was further confirmed by sequencing.

The pSVSPORT and pSVSPORT-L-methioninase expression plasmids are electro-transformed to VNP20009 strain (YS1646, ATCC No. <NUM>) respectively, and named as VNP20009-V and VNP20009-M, respectively. The specific construction process is as follows:
The competent bacteria VNP20009 was placed on ice, after melted, transferred to a pre-cooled electric rotating-cup and <NUM> of the plasmid was added, mixed well by flicking, incubated on ice for <NUM>; after heat shock <NUM> at <NUM>, placed on ice for <NUM>. The electric rotating-cup was placed into an electroporator, and the condition was set to voltage 2400V, resistance 400Ω, capacitance 25µF, discharge time <NUM>. After the electric shock, <NUM> SOC medium was added and mixed well gently, incubated at <NUM> for <NUM> with shaking. After the bacterial precipitation was blown by a pipette and uniformly spread on an ampicillin- resistant LB-O medium plate, then incubated <NUM> at <NUM> incubator. After the VNP20009-V and VNP20009-M were cultured with LB-O, the plasmids were extracted and identified by restriction enzyme digestion.

The protein was extracted from <NUM>×<NUM><NUM> Salmonella and separate by <NUM>% SDS-PAGE electrophoresis, transferred to PVDF membrane under constant voltage, after blocked <NUM> with BSA at room temperature, rinsed <NUM> × <NUM> with TBST, added with the rabbit anti-L-methioninase antibody (<NUM>: <NUM>) overnight at <NUM>, rinsed with TBST <NUM> times, <NUM> each time, then HRP labeled anti-rabbit secondary antibody (<NUM>: <NUM>) was added, incubated at room temperature for <NUM>, rinsed with TBST <NUM> times, <NUM> each time, developed using the enhanced chemiluminescent (ECL). The results are shown in <FIG>. Specific bands were found at about <NUM> kD molecular weight, indicating that the expression of L-methioninase significantly increased in VNP20009-M compared with that in VNP20009 and VNP20009-V.

L-methionine and pyridoxal were mixed with VNP20009-V and VNP20009-M strains respectively, and incubated at <NUM> for <NUM>. After terminated by <NUM>% trichloroacetic acid, the mixed solution was centrifuged to get the supernatant, then well mixed with <NUM>-methyl-<NUM> MBTH; after incubated at <NUM> for <NUM>, the absorbance at <NUM> was determined. The amount of enzyme that catalyzes to covert α-ketobutyric acid was defined as one unit of enzyme activity. The results are shown in <FIG>. The methioninase activity of Salmonella VNP20009-M was <NUM> times higher than that of VNP20009-V.

The above animal tests further showed that the attenuated Salmonella typhimurium VNP20009-M used in the present invention can inhibit tumor metastasis by inhibiting the expression and activity of EZH2, and has a good antitumor effect.

Claim 1:
A genetically engineered strain VNP20009-M for use in preventing or treating metastasis of a cancer in a human subject, wherein the genetically engineered strain VNP20009-M is an attenuated Salmonella typhimurium VNP20009 cloned with an L-methioninase gene, and wherein the cancer is prostate cancer, pancreatic cancer, breast cancer, liver cancer or leukemia.