Applications of genetically engineered bacteria VNP20009-M in preparation of drugs for preventing and treating lung cancer

Abstract

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

Claims

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

2. The method of claim 1, wherein the lung cancer is a primary lung tumor, a recurrent tumor after lung cancer surgery or a metastatic tumor from lung cancer.

3. The method of claim 1, wherein the lung cancer is non-small lung cancer or small cell lung cancer.

4. The method of claim 3, wherein the non-small cell lung cancer is squamous carcinoma, adenocarcinoma, adenosquamous carcinoma, large cell lung cancer or undifferentiated carcinoma.

5. The method of claim 1, wherein the genetically engineered bacterium is administered at a dose of at least 3.5×10.sup.7 CFU/M.sup.2.

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

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

8. The method of claim 7, wherein the genetically engineered bacterium is administered via intratumoral injection.

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

10. 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.

11. The method of claim 1, wherein the genetically engineered bacterium is constructed by: subcloing the L-methioninase gene into the plasmid, then electro-transforming 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 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 a condition of new lesions in the neck of the patient in Example 2;

(6) FIG. 5 shows a biopsy cell smear of the patient's tumor in Example 2;

(7) FIG. 6 shows a condition of the tumor of the patient after 3 weeks of treatment in Example 2;

(8) FIG. 7 shows a condition of the mass of the patient after 5 weeks of treatment in Example 2;

(9) FIG. 8 shows a condition of the original lesion site of the patient after 12 weeks of treatment in Example 2;

(10) FIG. 9 is a cytological smear of the patient's tumor after 1 week of treatment in Example 2;

(11) FIG. 10 is a cytological smear of the patient's tumor after 3 weeks of treatment in Example 2.

DETAILED DESCRIPTION

Example 1 Construction of Genetically Engineered Bacteria VNP20009-M

(12) 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.

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

(14) 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:

(15) 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.

(16) 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.

(17) 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.

(18) The ligation product was transformed into competent cells of E. coli DH5a (Takara). A tube of 50 μL of DH5a competent cells was placed on ice. After the ice was melt, 5 μL of the above-mentioned ligation product was added into the DH5a 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.

(19) 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.

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

(21) 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:

(22) 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.

(23) 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.

(24) 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.

(25) 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 Antitumor Effect of Genetically Engineered Bacterium VNP20009-M

(26) 1) Past Medical History and Diagnosis

(27) After a 73 years old male patient underwent a thoracoscopic right lung lower lobe squamous cell carcinoma radical operation for 5 months, a new mass was found in the neck (as shown in FIG. 4). The size of the lesion mass at the neck was measured as about 8 cm*9 cm. Cancer cells were identified by a cell smear from the lesion site biopsy (as shown in FIG. 5). In addition, a bone ECT showed multiple active bone metabolism; and a chest CT examination report showed sternal destruction with mass formation.

(28) According to the previous treatments and related examinations, the patient was diagnosed as recurrence and metastasis after the right lung lower lobe squamous cell carcinoma operation, but there was no standard treatment plan in clinic.

(29) 2) Treatment Plan

(30) The diluted VNP20009-M was evenly injected into the tumor site in multi-points and during the first time, 6*10.sup.7 cfu (about 3.5*10.sup.7 cfu/m.sup.2) of the VNP20009-M was administered. After a one-week interval, a second administration was conducted and a total amount of the drug was increased to 9*10.sup.7 cfu (about 5*10.sup.7 cfu/m.sup.2). An intratumoral injection was conducted by even and multi-point drug injection. After a one-week interval, a third administration was conducted with the same method and dose as in the 2.sup.nd administration. After a 10-day interval, a fourth administration was conducted. The drug concentration was increased to 6*10.sup.7 cfu/m.sup.2, and the intratumoral administration was conducted. After a 10-day interval, a fifth administration was conducted with the same method and dose as the 4th administration. A specific implementation plan is shown in Table 1 below.

(31) TABLE-US-00001 TABLE 1 Treatment implementation plan Times Time Dose 1.sup.st 0 day 3.5*10.sup.7 cfu/m.sup.2 2.sup.nd 7.sup.th day 5*10.sup.7 cfu/m.sup.2 3.sup.rd 14.sup.th day 5*10.sup.7 cfu/m.sup.2 4.sup.th 24.sup.th day 6*10.sup.7 cfu/m.sup.2 5.sup.th 34.sup.th day 6*10.sup.7 cfu/m.sup.2
3) Efficacy
3.1 Changes of Lesion Sizes

(32) The size of the lesion mass at the neck was measured to be approximately 8 cm*9 cm before the treatment (as shown in FIG. 4). After 3 weeks of the treatment, the mass was significantly reduced (as shown in FIG. 6). After 5 weeks of the treatment (the end of the 5-times treatments), the mass basically disappeared (as shown in FIG. 7). After 12 weeks of the treatment, there was no abnormal change in the neck clavicle, i.e., the original lesion site (as shown in FIG. 8).

(33) 3.2 Changes Inside the Tumor

(34) Before the treatment, the inside of the mass was of a cystic structure. And more severely abnormal-shaped cells, i.e., tumor cells were found by a cytological smear analysis (as shown in FIG. 5).

(35) One week after the treatment (one administration), the mass was significantly softened (as shown in FIG. 9). A large number of neutrophils and a small number of tumor cells were found by a cytological smear. Ten days after the treatment (with two administrations), about 40 mL of effusion was extracted. Two weeks after the treatment (twice treatment), the inside of the mass was further liquefied and about 90 mL of the effusion was extracted. Three weeks after the treatment (with three administrations), the tumor was shrunk (as shown in FIG. 10). About 45 mL of the effusion was continuously extracted. A large number of inflammatory cells and a very small number of tumor cells were found by a cytological smear. It was observed that the inflammatory cells wrapped around the periphery of the tumor cells. One month after the treatment (with four administrations), the tumor was further reduced; and the extracted effusion was reduced to about 20 mL. After the end of the fifth administration, no effusion was detected, and the mass was eliminated.

(36) The above results indicate that the injection of the genetically engineered bacteria VNP20009-M of the present invention into the inside of the tumor lesion induced local inflammatory cell infiltration, thereby killing the tumor cells.

(37) 3.3 Side Effects

(38) On the day of each administration and 9-10 hours after the injection, the patient had a fever of about 38° C. and restored to normal body temperature by physical cooling. On the day of administration, nausea and vomiting sometimes occurred for about 10 minutes. Other than that, there was no abnormal feeling. During the treatment, various indicators of liver and kidney functions were examined; and the results are shown in Table 2 below. The results show that the various indicators of the liver and kidney functions of the patient are all in the normal range. The above results indicate that the VNP20009-M has no obvious toxicity to the human body.

(39) TABLE-US-00002 TABLE 2 Results of various examination indicators of the patient 1 week 5 weeks 12 weeks Before the after the 2 weeks after after the after the Indicators treatment treatment the treatment treatment treatment Reference value Alanine 22.1 25.9 38.7 23.3 18.1 0-60.0 U/L aminotransferase Aspartate 14.2 13.8 16 16.2 15 0-40.0 U/L aminotransferase Total bilirubin 10.22 7.98 8.52 8.68 11.14 2.00-20.50 μmol/L Alkaline 98.3 74.2 100.8 110.9 108.3 45-125 U/L phosphatase Albumin 36.6 35.5 37.18 39.8 44.2 40.00-55.00 g/L Urea 4.78 3.65 4.93 5.84 4.75 1.70-8.30 mmol/L Creatinine 61 65.2 48 48.8 53.7 42.0-104.0 μmol/L Blood platelet 352 361 406 296 281 100-300 10 * 9/L Sodium 132 130.3 134.6 133 135 137-147 mmol/L

(40) The above data prove that the genetically engineered bacterium VNP20009-M of the present invention can treat lung cancer, effectively killing the lung squamous cancer cells and eliminating the tumor lesions, and having no obvious toxic and side effects on the human body.

(41) It is apparent that the above-described examples are merely illustrative and are not intended to limit the invention. 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.