Application of genetically engineered bacterium expressing methionine enzyme and anti-tumor medicine in combined preparation of medicine for treating malignant tumors

Abstract

The invention provides application of a genetically engineered bacterium expressing methionine enzyme and an anti-tumor drug in combined preparation of a drug for treating malignant tumors. Through combined use of the two drugs, the effects of synergistically promoting tumor cell apoptosis and inhibiting tumor growth are achieved, the effect of SGN1 and chemotherapeutic drugs on treating malignant tumors is further enhanced, the anti-tumor effect of the drugs is enhanced, and the range of applicable patients is widened; meanwhile, the toxic and side effects of the medicine are reduced due to the relatively low dosage, and the safety of the SGN1 and the cis-platinum is further improved.

Description

Field of the Invention

[0001] The present application belongs to the field of anti-tumor. Specifically, the present application provides the use of genetically engineered bacteria expressing methioninase and anti-tumor drugs in the combined preparation of drugs for treating malignant tumors. Background Art

[0002] Osteosarcoma is a rapidly growing malignant tumor that occurs frequently in adolescents and the elderly. It is one of the most common primary malignant bone tumors. The peak age of onset is 10-14 years old, with the second peak occurring in people over 60 years old. The 5-year survival rate for osteosarcoma patients without metastasis at initial diagnosis is between 40% and 75%. Once the tumor recurs, the survival rate drops significantly. Approximately 10% to 20% of patients present with distant metastasis at their first visit, 90% of which are lung metastases. Once a patient's osteosarcoma develops distant metastasis, their 5-year survival rate plummets to 20% to 30%. Local recurrence and distant metastasis are the most important causes of death in osteosarcoma patients and are currently the main challenges in osteosarcoma treatment.

[0003] Osteosarcoma patients are typically treated with amputation to radically remove the lesion. However, this treatment approach has not significantly improved long-term survival. The concept of neoadjuvant chemotherapy was proposed in the 1970s. This treatment approach, combined with extensive tumor resection, has been used clinically, significantly improving the 5-year survival rate and limb salvage rate of osteosarcoma patients. Currently, systemic treatment for osteosarcoma primarily involves high-dose chemotherapy. The three most commonly used chemotherapy drugs are adriamycin (ADM), cisplatin (DDP), and high-dose methotrexate (MTX). In addition, there are reports suggesting that ifosfamide (IFO) is also effective in osteosarcoma. Although gene-targeted therapy and immunotherapy have garnered significant attention in recent years, these treatment options are primarily used as adjuvant therapy for patients with advanced recurrent tumors who have failed chemotherapy and have not been able to effectively prolong survival.

[0004] Due to the severity of the disease course in advanced cancers, low-dose chemotherapy is ineffective. While increasing the dose ensures efficacy, severe drug reactions often harm the patient. This suggests that the chemotoxicity of chemotherapy drugs determines their narrow therapeutic window, making them prone to adverse reactions in patients, a major challenge in their clinical use. Common adverse reactions to chemotherapy drugs include severe bone marrow suppression and multi-organ lesions, including those in the urinary and digestive systems. Due to the limited number of chemotherapy drugs available clinically, the bottleneck of drug selection also poses a current treatment bottleneck for some tumors.

[0005] Research has confirmed that methionine (Met) dependence is a common characteristic of most tumor cells, including breast, lung, colon, kidney, bladder, melanoma, and glioma, whereas normal cells do not exhibit Met dependence. Methioninase (L-methioninase) specifically breaks down Met into α-ketobutyrate, methanethiol, and ammonia gum, thereby reducing methionine levels in the body. Treatments targeting methionine primarily include dietary restriction and methioninase injection. While dietary restriction can slow tumor cell proliferation, long-term methionine restriction can cause malnutrition and metabolic disorders, and can also exacerbate cancer progression due to prolonged DNA hypomethylation. Methioninase injection can inhibit tumor growth. Clinical trials have shown that intravenous methioninase can also significantly reduce plasma methionine levels. However, since mammals do not naturally express methioninase, exogenous methioninase can provoke an immune response. Consequently, these issues limit the clinical application of both methionine-based therapies.

[0006] Research using bacteria as gene therapy vectors has made considerable progress, with mainstream vectors including Salmonella, Escherichia coli, and Listeria monocytogenes. SGN1 is a novel anti-tumor drug developed through genetic engineering, using attenuated Salmonella enterica serovar Typhimurium (VNP20009) as a vector to express L-methioninase, thereby inhibiting tumor growth and metastasis. VNP20009 is an attenuated Salmonella typhimurium strain with deletions of the msbB and purI genes. It is genetically stable and sensitive to antibiotics. The msbB gene is essential for lipid acylation in endotoxins; its deletion prevents acylation of the lipid A terminus, reducing the toxicity of the vector. The purI gene is involved in purine metabolism; its deletion requires exogenous adenine for bacterial growth. VNP20009 also reduces the body's own production of tumor necrosis factor (TNF), mitigating the inflammatory response induced by VNP20009. Therefore, the low pathogenicity of VNP20009 improves its safety for clinical treatment and lays a solid foundation for the safety and efficacy of SGN1 in clinical applications. SGN1, which overexpresses methioninase and can specifically deplete methionine in tumor tissue, has been widely used in cancer therapy research. Its anti-tumor effects have been demonstrated in a variety of solid tumors, including breast cancer (Patent No. ZL201310062253.7), pancreatic cancer (Patent No. ZL201310688936.3), prostate cancer (Patent No. ZL201410183149.8), liver cancer (Patent No. 201510546063.1), and malignant sarcoma (Patent No. 201710216811.9). Phase I clinical trials are underway in the United States (IND Nos. 026901 and 027295) and China. SGN1 is highly targeted and accumulates at tumor sites. Researchers have found in mouse models of various solid tumors that SGN1 is 200-1,000 times more abundant in tumors than in major organs like the liver. SGN1 preferentially accumulates and multiplies in hypoxic, necrotic areas of tumor tissue, directly killing tumor cells. The nonspecific inflammatory response induced by SGN1 can activate anti-tumor immune responses to kill tumors. Summary of the Invention

[0007] On the one hand, the present application provides the use of genetically engineered bacteria expressing a methioninase gene and an anti-tumor drug in the combined preparation of a drug for treating malignant tumors.

[0008] Furthermore, the genetically engineered bacteria expressing the methioninase gene is an attenuated Salmonella typhimurium that has been genetically engineered and carries a plasmid expressing the methioninase gene.

[0009] Furthermore, the methioninase gene expression plasmid is a pSVSPORT plasmid, a pTrc99A plasmid, a pcDNA3.1 plasmid, a pBR322 plasmid or a pET23a plasmid containing the methioninase gene.

[0010] Furthermore, the Salmonella typhimurium is VNP20009.

[0011] Furthermore, the genetically engineered bacteria expressing the methioninase gene is the genetically engineered bacteria SGN1.

[0012] The genetically engineered bacteria SGN1 is also known as VNP20009-M. Its specific preparation method is described in the patents entitled “A genetically engineered bacterium for treating breast cancer and its construction method and application” (Patent No. ZL201310062253.7), “Application of attenuated Salmonella typhimurium and its genetically engineered bacteria in the preparation of drugs for treating pancreatic cancer” (Patent No. ZL201310688936.3), and “Application of attenuated Salmonella typhimurium and its genetically engineered bacteria in the preparation of drugs for treating prostate cancer” (Patent No. ZL201310688936.7). L201410183149.8), the patent name is "Application of attenuated Salmonella typhimurium genetically engineered bacteria in the preparation of drugs for the treatment of liver cancer" (patent number 201510546063.1), and the application name is "Application of genetically engineered bacteria VNP20009-M in the preparation of drugs for the treatment of malignant sarcoma" (application number CN201710216811.9), etc. Those skilled in the art can make routine adjustments to the preparation process and methioninase sequence on the basis of the above preparation methods and obtain the same or similar effects.

[0013] Furthermore, the anti-tumor drug is a chemotherapy drug.

[0014] Furthermore, the anti-tumor drug is cisplatin.

[0015] Furthermore, the malignant tumor is osteosarcoma.

[0016] Furthermore, the medicine is an injection.

[0017] Furthermore, the drug is an intratumoral injection drug, an intravenous injection drug or an interventional perfusion drug.

[0018] Furthermore, the drug reduces tumor volume and / or weight, increases tumor cell apoptosis and / or inhibits tumor cell proliferation.

[0019] On the other hand, the present application provides a drug for treating malignant tumors, which comprises a genetically engineered bacterium expressing a methioninase gene and an anti-tumor drug.

[0020] Furthermore, the genetically engineered bacteria expressing the methioninase gene is an attenuated Salmonella typhimurium that has been genetically engineered and carries a plasmid expressing the methioninase gene.

[0021] Furthermore, the methioninase gene expression plasmid is a pSVSPORT plasmid, a pTrc99A plasmid, a pcDNA3.1 plasmid, a pBR322 plasmid or a pET23a plasmid containing the methioninase gene.

[0022] Furthermore, the Salmonella typhimurium is VNP20009.

[0023] Furthermore, the genetically engineered bacteria expressing the methioninase gene is the genetically engineered bacteria SGN1.

[0024] Furthermore, the anti-tumor drug is a chemotherapy drug.

[0025] Furthermore, the anti-tumor drug is cisplatin.

[0026] Furthermore, the malignant tumor is osteosarcoma.

[0027] Furthermore, the medicine is an injection.

[0028] Furthermore, the drug is an intratumoral injection drug, an intravenous injection drug or an interventional perfusion drug.

[0029] The genetically engineered bacteria expressing the methioninase gene and the antitumor drug in the drug can be packaged separately and then combined in the same larger package, or they can be packaged separately and provided separately, and the corresponding instructions can indicate how to administer the two together.

[0030] On the other hand, the present application provides a method for treating malignant tumors by combining genetically engineered bacteria expressing the methioninase gene with anti-tumor drugs.

[0031] Furthermore, the genetically engineered bacteria expressing the methioninase gene is an attenuated Salmonella typhimurium that has been genetically engineered and carries a plasmid expressing the methioninase gene.

[0032] Furthermore, the methioninase gene expression plasmid is a pSVSPORT plasmid, a pTrc99A plasmid, a pcDNA3.1 plasmid, a pBR322 plasmid or a pET23a plasmid containing the methioninase gene.

[0033] Furthermore, the Salmonella typhimurium is VNP20009.

[0034] Furthermore, the genetically engineered bacteria expressing the methioninase gene is the genetically engineered bacteria SGN1.

[0035] Furthermore, the anti-tumor drug is a chemotherapy drug.

[0036] Furthermore, the anti-tumor drug is cisplatin.

[0037] Furthermore, the malignant tumor is osteosarcoma.

[0038] Furthermore, the genetically engineered bacteria SGN1 is administered by injection.

[0039] Furthermore, the genetically engineered bacteria SGN1 is administered by intratumoral injection, intravenous injection or interventional perfusion.

[0040] Furthermore, the genetically engineered bacteria expressing the methioninase gene and the anti-tumor drug are administered simultaneously or sequentially within a certain period of time.

[0041] Furthermore, the genetically engineered bacteria SGN1 was administered intravenously once a week at a dose of 4-9*10^8 CFU / time; intratumorally once a week for 3 consecutive weeks followed by a 1-week rest at a dose of 2-6*10^8 CFU / time; and intra-arterial infusion was administered once every 2 weeks at a dose of 4-9*10^8 CFU / time for long-term use.

[0042] Furthermore, cisplatin 50-100 mg / m2, once every 3 weeks, is used long-term.

[0043] The present invention discloses the application of a combined strategy of genetically engineered bacteria and chemotherapy drugs in the treatment of malignant tumors. The genetically engineered bacteria, SGN1, is an attenuated Salmonella typhimurium cloned with the methioninase gene. This genetically engineered bacterium can continuously express L-methioninase in tumor tissue, consuming large amounts of methionine and other nutrients, arresting the cell cycle and promoting apoptosis, ultimately leading to nutrient deprivation and necrosis of tumor cells. SGN1 can also increase the sensitivity of tumor cells to chemotherapy drugs, reducing the dosage of chemotherapy drugs that kill tumor cells and improving the safety of anti-tumor treatment. By combining the two drugs, the present invention achieves a synergistic effect of promoting tumor cell apoptosis and inhibiting tumor growth, further enhancing the efficacy of SGN1 and chemotherapy drugs in treating malignant tumors, thereby enhancing the anti-tumor effect of the drugs and broadening the range of patients suitable for treatment.

[0044] The present invention provides a strategy for combining a biopharmaceutical with a chemotherapy drug for the treatment of malignant tumors. Strategy 1: The combined dose of SGN1 and cisplatin is lower than the dose of either alone, reducing drug toxicity and side effects while enhancing therapeutic efficacy. This further improves the safety of SGN1 and cisplatin, resulting in a safer, more potent anti-tumor treatment strategy. It also reduces treatment risks and costs, meeting the treatment needs of malignant tumor patients and possessing promising application prospects. Strategy 2: SGN1 is used first to increase the sensitivity of tumor cells to chemotherapy drugs, allowing chemotherapy-resistant tumor patients to opt for chemotherapy treatment. This provides drug-resistant tumor patients with the opportunity to receive effective anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This figure shows the results of changes in the number of osteosarcoma cells after treatment with genetically engineered bacteria SGN1 and the chemotherapy drug cisplatin in vitro.

[0046] Figure 2 This figure shows the results of changes in the apoptosis ratio of osteosarcoma cells after treatment with genetically engineered bacteria SGN1 and the chemotherapy drug cisplatin in vitro.

[0047] Figure 3 This figure shows the results of the synergistic effect of genetically engineered bacteria SGN1 and the chemotherapy drug cisplatin on the proliferation inhibition of osteosarcoma cells.

[0048] Figure 4 This is the curve of osteosarcoma volume change after intratumoral injection of engineered bacteria SGN1 and intraperitoneal injection of the chemotherapy drug cisplatin.

[0049] Figure 5 The following diagram shows the tumors obtained after tumor removal in tumor-bearing nude mice of different treatment groups when the tumor volume of the PBS control group was greater than 1500 mm3.

[0050] Figure 6 This is a statistical analysis of the weight of the tumor obtained after tumor removal in tumor-bearing nude mice after injection of the engineered bacteria SGN1 and the chemotherapy drug cisplatin.

[0051] Figure 7 This is the result curve of the weight change of tumor-bearing nude mice when the tumor volume of the PBS control group is greater than about 1500 mm3.

[0052] Figure 8 This is the HE staining result of tumor tissue.

[0053] Figure 9 This is a statistical chart showing the changes in serum biochemical indicators in tumor-bearing nude mice after injection of engineered bacteria SGN1 and the chemotherapy drug cisplatin.

[0054] Figure 10This is the curve of osteosarcoma volume change after intravenous injection of engineered bacteria SGN1 and intraperitoneal chemotherapy drug cisplatin.

[0055] Figure 11 The figure shows the tumors obtained after tumor removal in tumor-bearing nude mice of different treatment groups when the tumor volume of the PBS control group was about 1500 mm3.

[0056] Figure 12 This is a statistical analysis of the weight of the tumor obtained after tumor removal in tumor-bearing nude mice after injection of the engineered bacteria SGN1 and the chemotherapy drug cisplatin.

[0057] Figure 13 This is the result graph of the weight change curve of tumor-bearing nude mice when the tumor volume of the PBS control group is about 1500 mm3.

[0058] Figure 14 This is the HE staining result of tumor tissue. DETAILED DESCRIPTION

[0059] Example 1 Construction of genetically engineered bacteria SGN1

[0060] The L-methioninase gene was expressed through Kpn I and Hind The III restriction site was subcloned into the pSVSPORT plasmid to obtain an L-methioninase expression plasmid; the L-methioninase expression plasmid was electroporated into attenuated Salmonella typhimurium VNP20009 to obtain VNP20009 bacteria carrying a plasmid cloned with the L-methioninase gene (named SGN1 in the present invention); and the empty plasmid pSVSPORT was transferred into attenuated Salmonella typhimurium VNP20009 to obtain VNP20009 bacteria carrying the empty plasmid pSVSPORT (named VNP-V in the present invention). The detailed construction methods of SGN1 and VNP-V have been disclosed in the patents entitled “A Genetically Engineered Bacteria for Treating Breast Cancer and Its Construction Method and Application” (Patent No. ZL201310062253.7) and “Application of Attenuated Salmonella typhimurium and Its Genetically Engineered Bacteria in the Preparation of Drugs for Treating Pancreatic Cancer” (Patent No. ZL201 310688936.3), patent name “Application of attenuated Salmonella typhimurium and genetically engineered bacteria thereof in the preparation of drugs for treating prostate cancer” (patent number ZL201410183149.8), patent name “Application of attenuated Salmonella typhimurium genetically engineered bacteria in the preparation of drugs for treating liver cancer” (patent number 201510546063.1), application name “Application of genetically engineered bacteria VNP20009-M in the preparation of drugs for treating malignant sarcoma” (application number CN201710216811.9), etc., among which the VNP20009 bacteria carrying the plasmid cloned with the L-methioninase gene (named SGN1 in the present invention) is named VNP20009-M in the above patents or patent applications, and the VNP20009 bacteria carrying the empty plasmid pSVSPORT (named VNP-V in the present invention) is named VNP20009-V in the above patents or patent applications.

[0061] Example 2 Effect of Combining Genetically Engineered Bacteria with Chemotherapeutic Drugs on Inhibiting Osteosarcoma Cell Growth

[0062] (1) Osteosarcoma cells MNNG-HOS or U2OS (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM medium containing 10% fetal bovine serum, and the cell suspension was collected; the cell suspension concentration was adjusted to 1×105

[0063] cells / ml, add 1 ml of cell suspension to a 6-well plate, add culture medium to 2 ml, and culture in a 37°C, 5% CO2 incubator overnight.

[0064] (2) The genetically engineered bacteria SGN1 obtained in Example 1 was cultured in LB-0 medium. When the OD value was ≈ 0.8, the bacteria were collected and resuspended in PBS. The culture medium was removed by centrifugation at 8000 rpm for 5 min. The bacteria were washed twice with saline and resuspended in saline. The bacterial concentration was adjusted to 1 × 109 CFU / mL for subsequent bacterial cell co-culture.

[0065] (3) Weigh 3 mg of cisplatin powder (purchased from selleck) and dissolve it in 10 mL of sterile pure water. Dissolve it completely to prepare a cisplatin mother solution with a concentration of 1 mM. Then filter it through a 0.22 μM filter and aliquot it for use.

[0066] (4) Pipette 0.2 mL of 1 mM cisplatin stock solution and mix evenly with 1.8 mL of PBS to prepare a 100 μM cisplatin working solution for later use.

[0067] (5) Experimental grouping: ① control; ② DDP 1 μM; ③ SGN1 0.5 MOI; ④ SGN1 0.5 MOI + DDP 1 μM; ⑤ SGN1 1 MOI; ⑥ SGN1 1 MOI + DDP 1 μM; ⑦ SGN1 2.5 MOI; ⑧ SGN1 2.5 MOI + DDP 1 μM; ⑨ SGN1 5 MOI; ⑩ SGN1 5 MOI + DDP 1 μM; SGN1 10 MOI; SGN1 10MOI+DDP 1μM;

[0068] (6) After the plated cells have adhered to the wall in the incubator for 3 hours, the diluted SGN1 bacterial solution is added to the wells of the SGN1 group and the combination group, 100 μL per well. 100 μL of saline is added to the control and DDP groups, and the mixture is gently shaken for uniform distribution. The wells are cultured in a 37°C, 5% CO2 incubator for 5 hours. After 5 hours of culture, the culture medium is pipetted into a 10 mL EP tube, and the wells are rinsed twice with PBS containing 50x gentamicin. The culture medium is filtered using a 10 mL syringe and a 0.22 μM disposable syringe filter, and 40 μL of 50x gentamicin is added. Finally, the filtered culture medium is added back to the wells, 2 mL per well. The wells are cultured in a 37°C, 5% CO2 incubator for another 19 hours. After culturing for 24 hours, the corresponding volume of cisplatin solution is added to the cisplatin-treated wells. For the control and SGN1 alone treatment groups, 40 μL of saline is added. After adding the solution, shake gently to mix evenly, and place in an incubator at 37°C, 5% CO2 for another 48 hours.

[0069] (7) After 72 hours of cell culture, discard the culture medium in the wells, rinse twice with 1 mL of PBS, add 0.3 mL of 0.25% trypsin, and digest in a 37°C incubator for 3 minutes. Resuspend the cells in 2 mL of complete culture medium to prepare a cell suspension for counting. The counting results were processed according to the formulas "cell concentration = (cell count result × dilution factor × 104) / 4", "cell viability = cell concentration of experimental group / cell concentration of control group × 100%", and "inhibition rate = 1-cell viability" to calculate the cell inhibition rate after treatment for each drug group.

[0070] (8) Osteosarcoma cells were treated with drugs according to the above method. Osteosarcoma cells MNNG-HOS were grouped as follows: ① control; ② DDP 2μM; ③ SGN1 2.5MOI; ④ SGN1 2.5MOI + DDP 2μM. Osteosarcoma cells U2OS were grouped as follows: ① control; ② DDP 1μM; ③ SGN1 1MOI; ④ SGN1 1MOI + DDP 1μM. 72 hours after treatment, cells were collected by trypsin digestion and added to pre-cooled PBS buffer. Centrifugation was performed at 300×g for 5 minutes, and the supernatant was discarded. This was repeated twice. After centrifugation, PBS buffer was added to resuspend the cells, and 100μl of the cell suspension was aspirated and added to a flow cytometer. 5μl of FITC-Annexin V dye was added to the flow cytometer, gently vortexed to mix, and incubated at room temperature in the dark for 15 minutes. Cell apoptosis was detected using a flow cytometer.

[0071] (9) Osteosarcoma cells were treated with drugs and counted according to the aforementioned method, and the groups were set as follows: ① control; ② SGN1 1MOI + DDP 0.5μM; ③ SGN1 1MOI + DDP 1μM; ④ SGN1 1MOI + DDP 2μM; ⑤ SGN1 2.5MOI + DDP 0.5μM; ⑥ SGN1 2.5MOI + DDP 1μM; ⑦ SGN1 2.5MOI + DDP 2μM; ⑧ SGN1 5MOI + DDP 0.5μM; ⑨ SGN1 5MOI + DDP 1μM; ⑩ SGN1 5MOI + DDP 2μM. The CI values ​​were calculated using Compusyn software, and the synergistic effect of SGN1 and cisplatin in osteosarcoma cells was analyzed (CI>1, indicating an antagonistic effect; CI=1, indicating an additive effect; CI<1, indicating a synergistic effect).

[0072] The results are as follows Figure 1-3 As shown, Figure 1 This is a graph showing the changes in the number of osteosarcoma cells after treatment with genetically engineered bacteria SGN1 and the chemotherapy drug cisplatin in vitro; Figure 2 This is a graph showing the changes in the apoptosis ratio of osteosarcoma cells after treatment with genetically engineered bacteria SGN1 and the chemotherapy drug cisplatin in vitro; Figure 3 This is the result of the synergistic effect of genetically engineered bacteria SGN1 and chemotherapy drug cisplatin on the proliferation inhibition of osteosarcoma cells. Figure 1 It can be seen that the viability of osteosarcoma cells without any drug treatment is 100%, and the use of SGN1 alone can significantly inhibit the viability of osteosarcoma cells in a dose-dependent manner. When treated with 0.5MOI of SGN1 alone, the viability of osteosarcoma cells is about 80-90%, and when treated with 1MOI of SGN1, the viability of osteosarcoma cells is about 80%; when treated with 2.5MOI of SGN1, the viability of osteosarcoma cells is about 60-70%; when treated with 5MOI of SGN1, the viability of osteosarcoma cells is about 40-50%; when treated with 10MOI of SGN1, the viability of osteosarcoma cells is about 100%. The viability of osteosarcoma cells is about 30%; the viability of osteosarcoma cells when treated with 1μM cisplatin alone is about 70%; the viability of osteosarcoma cells when treated with 1μM cisplatin combined with 0.5MOI of SGN1 is about 50-60%, the viability of osteosarcoma cells when treated with 1μM cisplatin combined with 1MOI of SGN1 is about 50%; the viability of osteosarcoma cells when treated with 1μM cisplatin combined with 2.5MOI of SGN1 is about 40%; the viability of osteosarcoma cells when treated with 1μM cisplatin combined with 5MOI of SGN1 is about 20-30%; the viability of osteosarcoma cells when treated with 1μM cisplatin combined with 10MOI of SGN1 is about 20%. Figure 2 It can be seen that 2.5MOI of SGN1 or 2μM cisplatin treatment alone can cause an increase in the apoptosis rate of osteosarcoma cells MNNG-HOS (about 10% and 12% respectively), and the combined treatment of SGN1 and cisplatin can synergistically and significantly increase the apoptosis rate of osteosarcoma cells MNNG-HOS (about 24%); while 1MOI of SGN1 cannot cause an increase in the apoptosis rate of osteosarcoma cells U2OS, while 1μM cisplatin treatment alone can cause an increase in the apoptosis rate of osteosarcoma cells U2OS (about 4%), and the combined treatment of SGN1 and cisplatin can synergistically and significantly increase the apoptosis rate of osteosarcoma cells U2OS (about 6%). Figure 3 It can be seen that when SGN1 is combined with cisplatin to treat MNNG-HOS cells under conditions of multiple different dose combinations, the calculated CI values ​​are all less than 1, indicating that the combination of the two has a synergistic effect; when SGN1 is combined with cisplatin to treat U2OS cells under conditions of multiple different dose combinations (SGN1 1MOI+DDP1μM, SGN11MOI+DDP 2μM, SGN1 2.5MOI+DDP 0.5μM, SGN1 2.5MOI+DDP 1μM), the calculated CI values ​​are all less than 1, indicating that the combination of the two has a synergistic effect.

[0073] The above results indicate that genetically engineered attenuated Salmonella SGN1 expressing methioninase can increase the sensitivity of osteosarcoma cells to chemotherapy drugs, and that the combination of SGN1 and the chemotherapy drug cisplatin can synergistically enhance the induction of osteosarcoma cell apoptosis, indicating that the combination of SGN1 and the chemotherapy drug cisplatin can synergistically kill tumor cells.

[0074] Example 3 Effect of Genetically Engineered Bacteria (Intratumoral Injection) Combined with Chemotherapeutic Drugs in Inhibiting Osteosarcoma Tumor Growth

[0075] (1) Osteosarcoma MNNG-HOS cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM medium containing 10% fetal bovine serum, and the cell suspension was collected. The concentration of the cell suspension was adjusted with PBS solution, and the target concentration of the suspension was 2×107 cells / ml. The cell suspension was then subcutaneously inoculated into the right axilla of BALB / C nude mice (Guangdong Laidi Biomedical Research Institute Co., Ltd.) at a dose of 100 μl / mouse. The tumor-bearing mice were randomly divided into the following groups: normal saline control group, cisplatin (DDP, 3 mg / kg) group, low-dose SGN1 (2×104 CFU / mouse) group, high-dose SGN1 (2×105 CFU / mouse) group, cisplatin + low-dose SGN1 (2×104 CFU / mouse) group, and cisplatin + high-dose SGN1 (2×104 CFU / mouse) group.

[0076] (2) The genetically engineered bacteria SGN1 obtained in Example 1 were cultured in LB-O medium. When OD ≈ 0.6, the bacteria were collected and then resuspended in PBS. When the average volume of the tumor in the tumor-bearing mice reached about 80 mm3, the mice were treated with group administration according to the method described in (1). SGN1 was administered by intratumoral injection on the first and eighth days, and cisplatin was administered by intraperitoneal injection on the fourth and eleventh days, respectively. The control group was injected with the same volume of normal saline. After administration, the mice were observed regularly, and the tumor size was measured with a vernier caliper to calculate the tumor volume (volume = 0.52 × length × width ×

[0077] Width), draw the mouse tumor volume change curve, weigh the mice with a balance, and draw the mouse weight change curve. When the average tumor volume of the control group is greater than 1500mm3, the mouse tumor is removed, photographed, and weighed. Another tumor tissue is fixed with 4% paraformaldehyde fixative for 24 hours, embedded in paraffin, sectioned, and HE stained. Blood is collected from the mice, allowed to stand at room temperature for 30 minutes, centrifuged at 3000g for 15 minutes, and the upper serum is collected and sent to Beijing Prilai Gene Technology Co., Ltd. for detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CR) levels.

[0078] The results are as follows Figure 4-9 As shown, Figure 4This is the curve of osteosarcoma volume change after intratumoral injection of engineered bacteria SGN1 and intraperitoneal injection of cisplatin. Figure 5 This is a diagram showing the tumors obtained after tumor removal in nude mice bearing tumors when the tumor volume in the normal saline control group is greater than 1500 mm3. Figure 6 This is a statistical analysis of the weight of the tumors obtained after tumor removal in the nude mice bearing tumors in different treatment groups when the tumor volume of the normal saline control group is greater than 1500 mm3. Figure 7 This is the result of the effect of intratumoral injection of engineered bacteria SGN1 and intraperitoneal injection of cisplatin on the body weight of tumor-bearing mice. Figure 8 This is the HE staining result of tumor tissue. Figure 9 This is the result of the changes in liver and kidney biochemical indicators in tumor-bearing nude mice after treatment. Figure 4-6 As can be seen from Table 1, the tumors of mice in the normal saline control group grew rapidly, while the tumor growth of mice was not significantly inhibited after treatment with low-dose SGN1 and the chemotherapy drug cisplatin alone. After treatment with high-dose SGN1 alone, the tumor growth of mice was significantly inhibited, and the volume or weight of the tumors in the SGN1 and cisplatin combination group was significantly smaller than that in the SGN1 alone or cisplatin alone group. However, the tumor inhibitory effect of the high-dose SGN1 and cisplatin combination was not significantly different from that of the low-dose SGN1 and cisplatin combination. After treatment, the body weight of mice in the SGN1 alone, cisplatin alone, and low-dose SGN1 and cisplatin combination groups did not change significantly compared with the control group, while the body weight of mice in the high-dose SGN1 and cisplatin combination group decreased significantly ( Figure 7 ). The results of HE staining of tumor tissue are shown in Figure 8 ,from Figure 8 It can be seen that the coloring of the normal saline control group is continuous and uniform; while the coloring of the SGN1 group is uneven, with obvious purple-red areas, suggesting that abnormal nuclear staining has occurred in these areas. When magnified 20 times, it was found that the cells in the SGN1 treatment group were swollen, the nuclei were condensed, and the normal morphology had been lost, indicating that the tumor tissue was necrotic; the SGN1 and cisplatin combination group also showed uneven coloring, with a loose structure between the cells, suggesting that abnormal tumor tissue staining has occurred in these areas. When magnified 20 times, it was found that the cells were also swollen, the nuclei were condensed, and the normal morphology had been lost, indicating that the tumor tissue was necrotic. Figure 9It can be seen that the ALT level of normal mice is about 40U / L, AST is about 200U / L, ALT / AST is about 0.25, and CR is about 7.5μmol / L. The ALT, ALT / AST and CR levels of the cisplatin alone group decreased slightly, and the AST and CR levels of the SGN1 alone group (2×104CFU / mouse) decreased slightly. The ALT level of the SGN1 (2×104CFU / mouse) and cisplatin combination group increased slightly, and the CR level decreased slightly, but there was no significant difference compared with the control group, indicating that the combination of low-dose SGN1 and cisplatin has no significant toxic effect on the liver and kidneys.

[0079] Table 1 Inhibitory effect of intratumoral injection of SGN1 combined with cisplatin on osteosarcoma growth

[0080] Grouping Inhibition rate Con 0.00％ DDP 3mg / kg 16.63％ SGN1 2*10^4cfu 21.81％ SGN1 2×10^5cfu 34.97％ SGN1 2×10^4 cfu+DDP 3 mg / kg 59.93％ SGN1 2×10^5 cfu+DDP 3 mg / kg 63.94％

[0081] The above results show that the attenuated Salmonella SGN1 that has been genetically engineered to express methioninase combined with the chemotherapy drug cisplatin can inhibit the growth of osteosarcoma, and the inhibitory effect is significantly improved compared with the use of SGN1 or cisplatin alone, without causing significant toxic effects on the liver and kidneys.

[0082] Example 4: Effect of Genetically Engineered Bacteria (Intravenous Injection) Combined with Chemotherapeutic Drugs in Inhibiting Osteosarcoma Tumor Growth

[0083] (1) Osteosarcoma MNNG-HOS cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM medium containing 10% fetal bovine serum, and the cell suspension was collected. The concentration of the cell suspension was adjusted with PBS solution, and the target concentration of the suspension was 2×107 cells / ml. The cell suspension was then subcutaneously inoculated into the right axilla of BALB / C nude mice (Guangdong Laidi Biomedical Research Institute Co., Ltd.) at a dose of 100 μl / mouse. The tumor-bearing mice were randomly divided into the following groups: saline control group, cisplatin (DDP, 2 mg / kg) group, SGN1 (2×104 CFU / mouse) group, and cisplatin + SGN1 group.

[0084] (2) The genetically engineered bacteria SGN1 obtained in Example 1 was cultured with LB-O medium. When OD ≈ 0.6, the bacteria were collected and then resuspended with PBS. When the average volume of the tumor in the tumor-bearing mice reached about 80 mm3, the grouping and administration were carried out according to the method described in (1). SGN1 was administered by tail vein injection on the first and eighth days, and cisplatin was administered by intraperitoneal injection on the fourth and eleventh days. The control group was injected with the same volume of normal saline. After administration, the status of the mice was observed regularly, the tumor size was measured with a vernier caliper, and the tumor volume was calculated (volume = 0.52 × length × width × width), and the mouse tumor volume change curve was drawn ( Figure 10 ), weigh the mice with a balance, and draw the weight change curve of the mice ( Figure 11 When the average tumor volume of the control group was greater than 1500 mm3, the mouse tumor was removed, photographed, and weighed ( Figure 12 、 13 ).

[0085] The results are as follows Figure 10-14 As shown in Table 2, Figure 10 This is the curve of osteosarcoma volume change after intravenous injection of engineered bacteria SGN1 and intraperitoneal injection of cisplatin. Figure 11 This is a picture showing the tumor obtained after tumor removal in nude mice bearing tumors when the tumor volume of the normal saline control group was about 1500 mm3. Figure 12 This is a statistical analysis of the weight of the tumors obtained after tumor removal in the nude mice bearing tumors in different treatment groups when the tumor volume of the normal saline control group was about 1500 mm3. Figure 13 This is the result of the effect of intravenous injection of engineered bacteria SGN1 and intraperitoneal injection of cisplatin on the body weight of tumor-bearing mice. Figure 14 This is the HE staining result of tumor tissue. Figure 10-12 It can be seen that the tumors of mice in the normal saline control group grew rapidly, while the tumor growth of mice was not significantly inhibited after treatment with the chemotherapy drug cisplatin alone. After treatment with SGN1 alone, the tumor growth of mice was significantly inhibited, and the volume or weight of the tumors in the SGN1 and cisplatin combination group was significantly smaller than that in the SGN1 alone or cisplatin alone groups. After treatment, the weight of mice in the SGN1 alone group did not change significantly compared with the control group, while the weight of mice in the cisplatin alone group and the SGN1 and cisplatin combination group decreased by ( Figure 13 ). The results of HE staining of tumor tissue are shown in Figure 14 ,from Figure 14 As can be seen, the normal saline control group was stained continuously and evenly; while the SGN1 group was stained unevenly, with obvious purple-red areas, suggesting that abnormal nuclear staining occurred in these areas. When magnified 20 times, it was found that the cells in the SGN1 treatment group were swollen, the nuclei were condensed, and they lost their normal morphology, indicating that the tumor tissue was necrotic. The SGN1 and cisplatin combination group also showed uneven staining, with a loose structure between the cells, suggesting that abnormal tumor tissue staining occurred in these areas. When magnified 20 times, it was found that the cells were swollen, the nuclei were condensed, and they lost their normal morphology, indicating that the tumor tissue was necrotic.

[0086] Table 2 Inhibitory effect of intravenous injection of SGN1 combined with cisplatin on osteosarcoma growth

[0087] Grouping Inhibition rate Con 0.00％ DDP 2mg / kg 10.37％ SGN1 2*10^4cfu 25.41％ SGN1 2×10^4 cfu+DDP 2 mg / kg 47.46％

[0088] The above results show that the attenuated Salmonella SGN1 that has been genetically engineered to express methioninase can be used in combination with the chemotherapy drug cisplatin to inhibit the growth of osteosarcoma, and the inhibitory effect is significantly improved compared with the use of SGN1 or cisplatin alone.

Claims

1. The application of genetically engineered bacteria expressing methioninase and anti-tumor drugs in the combined preparation of drugs for the treatment of malignant tumors.

2. The use according to claim 1, wherein the genetically engineered bacteria expressing the methioninase gene is an attenuated Salmonella typhimurium that has been genetically engineered and carries a plasmid expressing the methioninase gene.

3. The use according to claim 2, wherein the methioninase gene expression plasmid is a pSVSPORT plasmid, a pTrc99A plasmid, a pcDNA3.1 plasmid, a pBR322 plasmid or a pET23a plasmid containing the methioninase gene.

4. The use according to claim 2 or 3, wherein the Salmonella typhimurium is VNP20009.

5. The use according to claim 1, wherein the genetically engineered bacterium expressing the methioninase gene is the genetically engineered bacterium SGN1.

6. The use according to any one of claims 1 to 5, wherein the anti-tumor drug is a chemotherapy drug.

7. The use according to claim 6, wherein the anti-tumor drug is cisplatin.

8. The use according to any one of claims 1 to 7, wherein the malignant tumor is osteosarcoma.

9. The use according to any one of claims 1 to 8, wherein the medicine is an injection.

10. The use according to claim 9, wherein the drug is an intratumoral injection drug, an intravenous injection drug, or an interventional perfusion drug.

11. The use according to any one of claims 1 to 10, wherein the drug reduces tumor volume and / or weight, increases tumor cell death and / or inhibits tumor cell proliferation.

12. A drug for treating malignant tumors, comprising a genetically engineered bacterium expressing a methioninase gene and an anti-tumor drug.

13. The drug according to claim 12, wherein the genetically engineered bacteria expressing the methioninase gene is an attenuated Salmonella typhimurium that has been genetically engineered and carries a plasmid expressing the methioninase gene.

14. The drug according to claim 13, wherein the methioninase gene expression plasmid is a pSVSPORT plasmid, a pTrc99A plasmid, a pcDNA3.1 plasmid, a pBR322 plasmid or a pET23a plasmid containing the methioninase gene. The medicine according to claim 13 or 14, wherein the Salmonella typhimurium is VNP20009. The drug according to claim 12 , wherein the genetically engineered bacterium expressing the methioninase gene is the genetically engineered bacterium SGN1. The drug according to any one of claims 12 to 16, wherein the anti-tumor drug is a chemotherapy drug. The drug according to claim 17 , wherein the anti-tumor drug is cisplatin. The medicine according to any one of claims 12 to 18, wherein the malignant tumor is osteosarcoma.

20. The medicine according to any one of claims 12 to 19, which is an injection. The drug according to claim 20 , wherein the drug is an intratumoral injection drug, an intravenous injection drug, or an interventional perfusion drug.

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