Application of genetically engineered bacteria VNP20009-M in preparing drug for treating malignant sarcoma

Abstract

Provided is application of genetically engineered bacteria VNP20009-M in preparation of drugs for preventing and treating malignant sarcoma.

Claims

1. A method for treating malignant sarcoma, the method comprising administering a therapeutically effective amount of genetically engineered bacterium to a human having malignant sarcoma, wherein the genetically engineered bacterium is attenuated Salmonella typhimurium VNP20009 cloned with a L-methioninase gene.

2. The method of claim 1, wherein the malignant sarcoma is derived from a mesenchymal tissue.

3. The method of claim 2, wherein the mesenchymal tissue is a connective tissue or muscle.

4. The method of claim 1, wherein the malignant sarcoma occurs at fat, fascias, muscles, fibers, lymph, blood vessels, periosteum, and both ends of long bones.

5. The method of claim 1, wherein the malignant sarcoma is a soft tissue sarcoma or bone sarcoma.

6. The method of claim 1, wherein the malignant sarcoma is a primary sarcoma, a postoperative recurrent sarcoma, or a sarcoma metastasized to other sites after surgery.

7. The method of claim 6, wherein the malignant sarcoma is a sarcoma metastasized to lungs after surgery.

8. The method of claim 1, wherein the genetically engineered bacterium is administered at a dose of at least 6.4×10.sup.7 CFU/M.sup.2.

9. The method of claim 1, wherein the genetically engineered bacterium is administered once a week.

10. The method of claim 1, wherein the genetically engineered bacterium is administered orally, locally or via injection.

11. The method of claim 10, wherein the genetically engineered bacterium is administered via transvenous injection, peritoneal injection, subcutaneous injection, intramuscular injection, or intratumoral injection.

12. The method of claim 1, wherein the genetically engineered bacterium carries a plasmid which is cloned with the L-methioninase gene.

13. The method of claim 1, wherein the plasmid is a pSVSPORT plasmid, a pTrc99A plasmid, a pcDNA3.1 plasmid, a pBR322 plasmid or a pET23a plasmid.

14. The method of claim 1, wherein the genetically engineered bacterium is constructed by: subcloing the L-methioninase gene into the plasmid, then electrotransforming the plasmid to attenuated Salmonella typhimurium VNP20009.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to make the content of the present invention easier to understand, the present invention will be further described in detail below with reference to the embodiments of the present invention accompanying with the drawings, wherein

(2) FIG. 1 is a diagram of 1% agarose gel electrophoresis to identify the plasmid pSVSPORT-L-methioninase by enzyme digestion;

(3) FIG. 2 is a diagram showing the results of methioninase expression identified by Western blot according to the present invention;

(4) FIG. 3 is a diagram showing the results of detecting methioninase activity in salmonella according to the present invention;

(5) FIG. 4 shows the chest CT examination results of a lesion condition of the patient before treatment in Example 2;

(6) FIG. 5 is the result of a chest CT examination of the patient after 4 weeks of the treatment in Example 2;

DETAILED DESCRIPTION

Example 1 Construction of Genetically Engineered Bacteria VNP20009-M

(7) The construction method and processes of the genetically engineered bacterium VNP20009-M of the present invention are described in the examples of the Chinese Patent No. CN105983103A.

(8) (1) Construction of Plasmid Expressing L-Methioninase Gene

(9) The L-methioninase (GenBank: L43133.1) gene was synthesized and subcloned into a pUC57 plasmid (GenScript). The pUC57 plasmid subcloned with the L-methioninase gene was then subcloned into a pSVSPORT plasmid (Invitrogen) by Kpn I and Hind III enzyme cutting sites to obtain a pSVSPORT-L-methioninase expression plasmid. The specific construction processes are as follows:

(10) the pSVSPORT plasmid was subjected to Kpn I and Hind III double enzyme cutting. An enzyme cutting system contained 2 μg of plasmid DNA, 3 μL of 10*buffer, 1.5 μL of Kpn I enzyme, 1.5 μL of Hind III enzyme, and ddH.sub.2O added to supplement sufficiently a volume to 30 μL, and incubated in warm bath at 37° C. for 3 h. Then the enzyme cutting system was separated by electrophoresis in 1% agarose gel. A DNA band of 4.1 kb was cut out and purified by a gel recovery and purification kit.

(11) The DNA fragment of a L-methioninase coding region was obtained by whole-gene synthesis. The obtained DNA fragment was subcloned into the pUC57 plasmid (GenScript). The pUC57 plasmid subcloned with the DNA fragment was subjected to the Kpn I and Hind III double enzyme cutting using an enzyme cutting system containing 3 μg of plasmid DNA, 3 μL of 10*buffer, 1.5 μL of Kpn I enzyme, 1.5 μL of Hind III enzyme, and ddH.sub.2O added to supplement sufficiently a volume to 30 μL, which was incubated in warm bath at 37° C. for 3 h. The enzyme cutting system was then separated by electrophoresis in 1% agarose gel. A DNA band of 1.2 kb was cut out and purified by a gel recovery and purification kit.

(12) The pSVSPORT (Kpn I/Hind III) and the DNA fragment of the L-methioninase coding region (Kpn I/Hind III) were ligated with a ligation reaction containing 2 μL of the vector, 6 μL of the inserting fragments and 1 μL of T4 DNA ligase and incubated in warm bath at 16° C. for 16 h.

(13) The ligation product was transformed into competent cells of E. coli DH5α (Takara). A tube of 50 μL of DH5α competent cells was placed on ice. After the ice was melt, 5 μL of the above-mentioned ligation product was added into the DH5α competent cells with slight flipping to mix. The mixture was incubated on ice for 30 min before heat shock at 42° C. for 60 s and then incubated on ice for 2 min. 500 μL of LB liquid medium without antibiotics was added to the mixture and incubated at 37° C. for 1 h with shaking, after which the material was spread on an ampicillin-containing LB culture medium plate and cultured overnight.

(14) After clones were grown, single colonies were picked into 3 mL of ampicillin-containing LB culture liquid, incubated in a shaker at 37° C. for 16 h. Plasmid DNA was extracted and identified by Kpn 1 and Hind 111 enzyme digestion. As shown in FIG. 1, the positive clone had two DNA bands of 4.1 kb and 1.2 kb. Sequencing confirmed that the sequences of the positive clones are completely correct.

(15) (2) Constructions of VNP20009 Bacterium Carrying a Plasmid and VNP20009 Bacterium Carrying a Plasmid Cloned with a L-Methioninase Gene

(16) pSVSPORT and pSVSPORT-L-methioninase expression plasmids were respectively electrotransformed into the VNP20009 bacterium strain (YS1646, ATCC No. 202165) which were respectively named as VNP20009-V and VNP20009-M. The specific construction processes are as follows:

(17) Competent bacteria VNP20009 were placed on ice, after the ice was melted, the competent bacteria VNP20009 were transferred to a pre-cooled electric rotating cup, 2 μL of the plasmid was added into the electric rotating cup, slight flipping and uniform mixing were conducted, and incubation was conducted on ice for 1 min. The electric rotating cup was put into an electric rotating instrument, and conditions were set as a voltage of 2,400 V, a resistance of 400Ω, a capacitance of 25 μF and a discharge time of 4 ms. 1 mL of a SOC culture medium was added immediately after electric shock, and gentle and even mixing was conducted. Shaking culture is conducted at 37° C. for 1 h; and after a pipettor was used to precipitate and blow the bacteria evenly, the bacteria were applied on an ampicillin-containing resistant LB-O culture medium plate. The plate was then put in an incubator for culture at 37° C. for 16 h. After the VNP20009-V and VNP20009-M were cultured with the LB-O, the plasmid was extracted and identified by enzyme cutting to be correct.

(18) 1×10.sup.8 of salmonella were taken, proteins were extracted by a protein lysate, 10% SDS-PAGE electrophoresis was conducted, then electrotransformation to a PVDF membrane in an ice bath under stable pressure was conducted, after BSA room temperature sealing was conducted for 1 h, TBST rinsing was conducted for 3*5 min, a rabbit anti-L-methioninase antibody was added (1:1000), and incubation was conducted overnight at 4° C. The TBST rinsing was conducted for 3 times with 5 min each time, then a HRP-labeled anti-rabbit secondary antibody (1:10000) was added, incubation was conducted at room temperature for 1 h, the TBST rinsing was conducted for 3 times with 5 min each time, and ECL chemiluminescence developing was conducted. The results are shown in FIG. 2, a specific band was observed at a molecular weight of about 43 kD, indicating that the expression level of the L-methioninase was significantly increased in the VNP20009-M compared with the VNP20009 and VNP20009-V.

(19) L-methionine and pyridoxal were respectively mixed with the VNP20009-V and VNP20009-M bacteria. After incubation was conducted at 37° C. for 10 min, termination was conducted with 50% trichloroacetic acid, centrifugation was conducted, a supernatant was taken, the supernatant was mixed fully and evenly with 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH), after incubation was conducted at 50° C. for 30 min, absorbance at 320 nm was measured, and the amount of enzyme used for catalytic conversion of 1 μmol of α-ketobutyric acid per minute was defined as 1 enzyme activity unit. The results show (as shown in FIG. 3) that the activity of the methioninase in the salmonella VNP20009-M was 10 times higher than that of VNP20009-V.

(20) Thus, the constructed genetically engineered salmonella VNP20009-M has a relatively high methioninase activity and can be used for preparation of a methioninase agent.

Example 2 Effect of Genetically Engineered Bacteria VNP20009-M for Treating Soft Tissue Sarcomas

(21) 1) Past Medical History and Diagnosis

(22) A clinical male patient, 64 years old, with a left leg mass, was identified as spindle cell sarcoma by needle biopsy at Nanjing Drum Tower Hospital. The patient was then subjected to tumor resection. The tissues were taken to be subjected to pathologic analysis, showing tumor cells SMA(−), DES (−), MyoDl (−), FN(+), STAT6 (−), CD34 (−), S100 (−), CKpan (−), EMA (−), Vimentin (++), in combination with HE slices; and the results were in line with malignant fibrous histiocytoma.

(23) A regular follow-up CT re-examination showed space-occupying right lung upper lobe soft tissues; and needle biopsy pathology showed the spindle cell sarcoma. According to the patient's past treatments and related examinations, recurrence of lung metastasis after the surgery of the spindle cell sarcoma was diagnosed.

(24) 2) Treatment Plan

(25) The genetically engineered bacteria VNP20009-M diluted with 250 mL of physiological saline were intravenously administered to the body at a dose of 6.4*10.sup.7 CFU/M.sup.2 at an interval of 1 week; and a total of 5 times of infusion was conducted.

(26) 3) Efficacy

(27) 3.1 Changes of Tumor Sizes

(28) A chest CT examination before the treatment showed a size of the right lung upper lobe lesion was approximately 18*10*10 cm (as shown in FIG. 4). After 4 weeks of the treatment, the CT examination showed that the size of the right lung upper lobe lesion was approximately 17*10*10 cm (as shown in FIG. 5), and there was no significant change in the tumor size compared with that before the treatment.

(29) 3.2 Changes Inside the Tumor

(30) Before the treatment, a CT image showed flaky liquefied necrosis inside the lung lesion. As shown in FIG. 4, the inside of the lesion circled by white lines presented uneven colors, the darker regions indicated that the cells here had become necrotic and the brighter regions indicated living cells. After 4 weeks of the treatment, the CT image showed a uniform structure inside the lesion (as shown in FIG. 5) with a CT value of approximately 10 HU. The CT value indicated substance density; and when the CT value was lower than 20 HU, the substance was liquid, suggesting that the tumor cells in the region were basically necrotic and presented liquid. Therefore, the tumor cells of the patient with the sarcoma were rapidly necrotic and liquefied after the treatment with the VNP20009-M. However, since the lesion was in the body, the liquid could not be extracted, so that shrinking of the lesion was not observed.

(31) 3.3 Side Effects

(32) On the day of each treatment, 5-6 hours after the infusion, the patient had a highest fever of about 39.6° C. and restored normal body temperature by physical cooling. Other than that, there was no other abnormal feeling. During the treatment, various indicators of liver and kidney functions were examined; and the results were shown in Table 1 below. Detection results showed that the various indicators of the patient body were basically similar to those before the treatment. The above results indicate that the VNP20009-M produced no extra toxic and side effects to the patient.

(33) TABLE-US-00001 TABLE 1 Various indicator data of the patient body 4 weeks 8 weeks Reference Before the 1 week after after the after the Indicators value treatment the treatment treatment treatment Alanine 0-33 U/L 20 26 16 19 aminotransferase Aspartate 0-32 U/L 16 20 13 21 aminotransferase Total bilirubin 0-21 μmol/L 8.3 9.5 11.7 15 Alkaline phosphatase 35-104 U/L 71 91 96 140 Lactic dehydrogenase 109-245 U/L 158 160 135 127 Albumin 35-52 g/L 27 30 29 24 Urea nitrogen 2.86-8.21 mmol/L 4.36 2.41 3.2 2.23 Creatinine 59-104 μmol/L 55 57 51 52 Potassium 3.5-5.1 mmol/L 3.1 4.14 4.01 3.56 Sodium 136-145 mmol/L 138 134 136 137 Blood platelet 125-350 118 153 155 103 10 * 9/L

(34) The above data prove that the VNP20009-M can effectively kill the malignant sarcoma cells and eliminate the tumor lesions, and has no severe toxic and side effects on the human body.

(35) It is apparent that the above-described embodiments are merely illustrative of the examples and are not intended to limit the embodiments. Other variations or modifications of the various forms may also be made by those of ordinary skill in the art in light of the above description. There is no need and no way to exhaust all of the embodiments. And the obvious variations or modifications derived therefrom are still in the protection scope created by the present invention.