WO2018177375A1 - 基因工程菌vnp20009-m在制备治疗恶性肉瘤药物中的应用 - Google Patents

基因工程菌vnp20009-m在制备治疗恶性肉瘤药物中的应用 Download PDF

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WO2018177375A1
WO2018177375A1 PCT/CN2018/081116 CN2018081116W WO2018177375A1 WO 2018177375 A1 WO2018177375 A1 WO 2018177375A1 CN 2018081116 W CN2018081116 W CN 2018081116W WO 2018177375 A1 WO2018177375 A1 WO 2018177375A1
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sarcoma
vnp20009
use according
genetically engineered
plasmid
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PCT/CN2018/081116
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French (fr)
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赵子建
林艳
周素瑾
李芳红
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广州华津医药科技有限公司
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Priority to KR1020217042303A priority Critical patent/KR20210158434A/ko
Priority to CA3056296A priority patent/CA3056296C/en
Priority to US16/497,825 priority patent/US11324781B2/en
Priority to AU2018241657A priority patent/AU2018241657B2/en
Priority to JP2019550212A priority patent/JP6917077B2/ja
Priority to KR1020197032492A priority patent/KR102344837B1/ko
Priority to EP18777971.5A priority patent/EP3583956A4/en
Publication of WO2018177375A1 publication Critical patent/WO2018177375A1/zh
Priority to ZA2019/06176A priority patent/ZA201906176B/en

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Definitions

  • the invention belongs to the technical field of genetic engineering drugs, and particularly relates to a new application of a genetic engineering bacteria VNP20009-M in preparing a medicament for preventing and treating malignant sarcoma.
  • Sarcoma is a malignant tumor derived from mesenchymal tissue (including connective tissue and muscle). It occurs mostly at the ends of fat, fascia, muscle, fiber, lymph and blood vessels, periosteum and long bones. Each sarcoma has different histology, biological characteristics and different local infiltration, hematogenous and lymphatic metastasis, of which sarcoma metastasis to the lung is more common.
  • sarcoma The clinical manifestation of sarcoma is a mass that develops when the mass enlarges the surrounding tissue.
  • the incidence of sarcoma is very low, with an annual incidence of 2.4-5 cases per 100,000 people, accounting for about 1% of adult malignancies, 15% of childhood malignancies, but accounting for 2% of all cancer-related mortality. Due to the numerous subtypes of sarcoma and the diverse biological behaviors, the diagnosis is difficult. It is often found that the disease is late, and about 1/3-1/2 of the patients die from sarcoma recurrence and metastasis.
  • the current medical treatment for the treatment of advanced sarcoma has been stagnant, chemotherapy is still the standard treatment, for example, the treatment with doxorubicin is the standard program, the total survival of the treated patients is only 12-16 months. Overall, the 5-year survival rate of early patients is about 60%-80%, and the 5-year survival rate of advanced patients is less than 20%.
  • methionine dependence is a characteristic of most tumor cells, which is manifested by the excessive demand for methionine in tumor cells, in the removal of methionine or by its precursor, homocysteine.
  • cell proliferation is inhibited; in the presence of methionine, it can grow normally, including prostate cancer, breast cancer, lung cancer and other more than ten kinds of malignant cells.
  • methionine dependence in normal cells.
  • Methods for causing methionine deficiency mainly include removing methionine from the diet or decomposing methionine using methioninase.
  • methioninase does not cause excessive metabolic problems and has an anti-tumor effect.
  • Salmonella is a group of Gram-negative, invasive intracellular facultative anaerobic bacteria parasitic in the gut of humans and animals.
  • the known strain VNP20009 is a carrier with high tumor targeting, safety and anti-tumor effect. It has significant tumor growth inhibition effects on various mouse solid tumor models such as malignant melanoma and lung cancer. Two Phase I clinical studies conducted in the United States have shown that they can be used in humans with safety, but no anti-tumor effects have been observed.
  • the technical problem to be solved by the present invention is to provide a new application of genetically engineered bacteria VNP20009-M in the preparation of a medicament for preventing and treating sarcoma.
  • the present invention discloses the use of the genetically engineered bacteria VNP20009-M for preparing a medicament for preventing and treating malignant sarcoma.
  • the sarcoma is a malignant sarcoma derived from mesenchymal tissue such as connective tissue and muscle.
  • the sarcoma comprises sarcomas occurring at the ends of fat, fascia, muscle, fiber, lymph and blood vessels, periosteum and long bones.
  • the sarcoma comprises soft tissue sarcoma and osteosarcoma.
  • undifferentiated pleomorphic sarcoma is the most common, accounting for 25-35%; followed by liposarcoma (LPS), accounting for 25-30%; leiomyosarcoma , LMS) 12%; synovial sarcoma (SS) accounted for 10%; malignant peripheral nerve sheath tumor (MPNST) accounted for 6%.
  • Osteosarcoma also known as osteosarcoma, occurs mostly in adolescents or children under the age of 20. Osteosarcoma is developed from the interstitial cell line, and the tumor grows rapidly due to the direct or indirect formation of tumor bone-like tissue and bone tissue through the cartilage stage. Osteosarcoma is most common in pediatric bone malignancies, about 5% of pediatric tumors.
  • the sarcoma includes primary sarcoma, postoperative recurrent sarcoma, or sarcoma that has metastasized to other sites after surgery.
  • the sarcoma is a tumor that is transferred to the lung after surgery for malignant sarcoma.
  • the genetically engineered bacteria VNP20009-M has a minimum effective administration dose of 6.4 ⁇ 10 7 CFU/m 2 .
  • the above-mentioned methods for the prevention and treatment of tumors can be administered by various routes including, but not limited to, oral administration, topical administration, and administration by injection (including but not limited to intravenous, peritoneal, subcutaneous, intramuscular, intratumoral). Administration) and the like.
  • the above-mentioned genetically engineered bacteria VNP20009-M of the present invention are known strains, and their properties, shapes and construction methods are as described in Chinese Patent No. CN105983103A.
  • the genetically engineered strain VNP20009-M is an attenuated Salmonella typhimurium VNP20009 cloned with the L-methioninase gene.
  • the genetically engineered strain VNP20009-M is an attenuated Salmonella typhimurium VNP20009 carrying a plasmid, wherein the plasmid has an L-methioninase gene cloned thereon.
  • the genetically engineered strain VNP20009-M was constructed as follows: the L-methioninase gene was subcloned into a plasmid to obtain an L-methioninase expression plasmid, and the Lmethioninase expression plasmid was electrotransformed into the attenuated Salmonella typhimurium VNP20009.
  • Such plasmids include, but are not limited to, pSVSPORT plasmid, pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid.
  • the L-methioninase gene is subcloned into a plasmid to obtain an L-methioninase expression plasmid, and then the Lmethioninase expression plasmid is electrotransformed to minus.
  • Genetically engineered bacteria were obtained from Salmonella typhimurium VNP20009.
  • the electrical conversion condition is a voltage of 2400V, a resistance of 400 ⁇ , a capacitance of 25 ⁇ F, and a discharge time of 4ms.
  • the invention also discloses the application of the genetic engineering strain VNP20009-M in preparing a methionine enzyme preparation.
  • the invention discloses a novel application of the genetic engineering bacteria VNP20009-M for treating malignant sarcoma on the existing basis, and the genetic engineering bacteria VNP20009-M can effectively kill tumor cells and eliminate tumor lesions, for primary sarcoma, Postoperative recurrence of tumors and tumor cells metastasized to other sites after malignant sarcoma have better killing effect and better therapeutic effect; especially for soft tissue sarcoma after metastasis to the lungs, it has better killing effect.
  • the effect; and the genetically engineered bacteria have no obvious toxic and side effects on the human body, and provide a safe and effective new way for the treatment of malignant sarcoma.
  • Figure 1 is a 1% agarose gel electrophoresis pattern of plasmid pSVSPORT-L-methioninase
  • FIG. 2 is a diagram showing the expression of methioninase by Western blot
  • Figure 3 is a graph showing the results of detecting methioninase activity in Salmonella according to the present invention.
  • Figure 4 is a view showing the condition of chest CT examination before treatment of the patient in Example 2;
  • Figure 5 is a graph showing the results of chest CT examination after 4 weeks of treatment in the patient of Example 2.
  • the L-methioninase (GenBank: L43133.1) gene was firstly subcloned into the pUC57 plasmid (Kinseri), and then subcloned into the pSVSPORT plasmid (invitrogen) by Kpn I and Hind III cleavage sites to obtain pSVSPORT-L- Methioninase expression plasmid.
  • the specific construction process is as follows:
  • the pSVSPORT plasmid was digested with Kpn I and Hind III.
  • the digestion system was: 2 ⁇ g of plasmid DNA, 3 ⁇ L of 10 ⁇ buffer, 1.5 ⁇ L of Kpn I enzyme, 1.5 ⁇ L of Hind III enzyme, and the volume of ddH2O was added to 30 ⁇ L, and the temperature was 37 ° C for 3 h. . Then, the digestion system was separated by electrophoresis on a 1% agarose gel, and a DNA band of 4.1 kb was cut out, and the DNA was purified using a gel recovery purification kit.
  • the DNA fragment of the L-methioninase coding region was subcloned into the pUC57 plasmid (Kingsui) by whole-genome synthesis, and digested with Kpn I and Hind III.
  • the digestion system was: 3 ⁇ g plasmid DNA, 3 ⁇ L 10 ⁇ buffer, 1.5 ⁇ L Kpn. I enzyme, 1.5 ⁇ L Hind III enzyme, add ddH2O to make up the volume to 30 ⁇ L, and warm at 37 ° C for 3 h. Then, the digestion system was separated by electrophoresis on a 1% agarose gel, and a 1.2 kb DNA band was excised, and the DNA was purified using a gel recovery purification kit.
  • pSVSPORT Kpn I/Hind III
  • L-methioninase coding region DNA fragment Kpn I/HindIII
  • the ligation product was transformed into competent cells of E. coli DH5 ⁇ (Takara). Take a tube of 50 ⁇ L of DH5 ⁇ competent cells and place them on ice. After they are thawed, add 5 ⁇ L of the above-mentioned ligation product, mix gently, then incubate on ice for 30 min; heat at 42 °C for 60 s, then let stand on ice. 2 min; 500 ⁇ L of non-resistant LB liquid medium was added, cultured at 37 ° C for 1 h, and then plated on an ampicillin-resistant LB medium plate, and cultured overnight.
  • the monoclonal colonies were picked into 3 mL of ampicillin-containing LB medium, and cultured at 37 °C for 16 h.
  • the plasmid DNA was extracted and identified by Kpn I and Hind III digestion.
  • the positive clones were 4.1 kb and 1.2 kb. The two DNA bands are shown in Figure 1. The sequence of the positive clone was further confirmed by sequencing to be completely correct.
  • VNP20009-V and VNP20009-M were each electroporated into VNP20009 strain (YS1646, ATCC No. 202165), designated as VNP20009-V and VNP20009-M, respectively.
  • the specific construction process is as follows:
  • the competent bacteria VNP20009 was placed on ice, and after it was melted, it was transferred to a pre-cooled electric rotor, 2 ⁇ L of the plasmid was added thereto, and the mixture was gently incubated and incubated on ice for 1 min. Put the electric rotor into the electro-rotation instrument, the condition is set to voltage 2400V, resistance 400 ⁇ , capacitance 25 ⁇ F, discharge time 4ms. Immediately after the electric shock, add 1 mL of SOC medium and mix gently. Incubate at 37 ° C for 1 h with shaking; the bacterial pellet was evenly pipetted with a pipette and plated on an ampicillin-resistant LB-O medium plate. The plates were then incubated for 16 h in a 37 ° C incubator. After VNP20009-V and VNP20009-M were cultured with LB-O, the plasmid was extracted and identified by enzyme digestion.
  • the genetically engineered Salmonella VNP20009-M has high methioninase activity and can be used as a preparation of a methionine enzyme.
  • a male patient, 64 years old, with a left leg mass was identified as spindle cell sarcoma by needle biopsy at Nanjing Gulou Hospital.
  • the pathology of the tissue was again analyzed, showing that the tumor cells were SMA(-), DES(-), MyoD1(-), FN(+), STAT6(-), CD34(-), S100(-), CKpan (-), EMA (-), Vimentin (++), combined with HE section, in line with malignant fibrous histiocytoma.
  • CT found that the soft tissue of the right upper lobe was occupied, and the biopsy pathology showed spindle cell sarcoma. According to the patient's previous treatment and related examinations, the recurrence of lung metastasis after spindle cell sarcoma was diagnosed.
  • the genetically engineered strain VNP20009-M diluted with 250 ml of physiological saline was intravenously administered to the body at a dose of 6.4 ⁇ 10 7 CFU/m 2 .
  • the infusion was performed 5 times at intervals of 1 week.
  • a chest CT scan before treatment showed that the lesion in the right upper lobe was about 18*10*10 cm (as shown in Figure 4). After 4 weeks of treatment, CT examination showed that the lesion size of the right upper lobe was about 17*10*10 cm (as shown in Figure 5), and there was no significant change in tumor size compared with before treatment.
  • CT images showed flaky liquefaction and necrosis inside the lung lesions.
  • the inside of the lesion of the white coil exhibits an uneven color, the darker area indicates that the cells here have been necrotic, and the brighter area indicates the living cells.
  • the CT image showed a uniform structure inside the lesion (as shown in Figure 5) with a CT value of approximately 10 HU.
  • the CT value indicates the density of the substance. When it is lower than 20HU, the substance is liquid, suggesting that the tumor cells in this area are basically necrotic and appear liquid. This indicates that the tumor cells of the sarcoma patients are rapidly necrotic and liquefied after treatment with VNP20009-M. However, because the lesion was in the body, fluid could not be drawn, so the lesion was not observed to shrink.
  • VNP20009-M can effectively kill malignant sarcoma cells, eliminate tumor lesions, and has no serious side effects on the human body.

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Abstract

提供了基因工程菌VNP20009-M在制备预防和治疗恶性肉瘤药物中的应用。

Description

基因工程菌VNP20009-M在制备治疗恶性肉瘤药物中的应用 技术领域
本发明属于基因工程药物技术领域，具体涉及一种基因工程菌VNP20009-M在制备预防和治疗恶性肉瘤药物中的新应用。
背景技术
癌症已经成为人类死亡的重要原因，2005年至2015年间癌症发病率增加了33％。世界卫生组织(WHO)发表的《全球癌症报告2014》预测全球癌症病例将呈现迅猛增长态势，由2012年的1400万人，逐年递增至2025年的1900万人，到2035年将达到2400万人。
肉瘤(Sarcoma)是来源于间叶组织(包括结缔组织和肌肉)的恶性肿瘤，多发生于脂肪、筋膜、肌肉、纤维、淋巴及血管、骨膜及长骨两端。每种肉瘤都有不同的组织学、生物学特性和不一样的局部浸润、血行和淋巴转移倾向，其中肉瘤向肺转移较为常见。
肉瘤的临床表现是肿块，当肿块增大压迫周围组织时产生症状。肉瘤的发病率很低，年发病率为2.4-5例/10万人，约占成人恶性肿瘤的1％，占儿童恶性肿瘤的15％，但却占所有癌症相关死亡率的2％。由于肉瘤亚型众多，生物学行为多种多样，因此诊断比较困难，往往发现时病情较晚，约1/3-1/2的患者死于肉瘤复发及转移。现有医疗手段对晚期肉瘤的治疗发展一直停滞不前，化疗仍然是标准的治疗方法，例如含阿霉素的治疗是标准方案，经治患者的总生存期大概只有12-16个月。总体来说，早期患者5年生存率约60％-80％，晚期患者5年生存率不足20％。
现有技术表明，甲硫氨酸依赖是绝大部分肿瘤细胞的特性，表现为肿瘤 细胞对甲硫氨酸的过分需求，在去除甲硫氨酸或以其前体--同型半胱氨酸替代的培养基培养时，细胞增殖受到抑制；而在甲硫氨酸存在的环境下则能正常生长，其中包括前列腺癌、乳腺癌、肺癌等十多种恶性肿瘤细胞。但是，正常细胞不存在甲硫氨酸依赖性。造成甲硫氨酸缺乏的方法主要包括去除膳食中的甲硫氨酸，或者利用甲硫氨酸酶分解甲硫氨酸。然而单独限制饮食中甲硫氨酸的摄入来降低甲硫氨酸水平效果有限，而长期限制甲硫氨酸的摄入会引起机体营养不良、代谢障碍。相比于饮食限制甲硫氨酸摄入，利用甲硫氨酸酶(methioninase)不会引发过度的代谢问题，并具抗肿瘤效应。
沙门菌属是一群在人和动物肠道内寄生的革兰氏阴性、侵袭性细胞内兼性厌氧菌。其中，已知菌株VNP20009是一种高肿瘤靶向性、安全性以及具有抗肿瘤效应的载体。它对恶性黑色素瘤、肺癌等多种小鼠实体瘤模型有显著的抑制肿瘤生长效果。美国进行的两项Ⅰ期临床研究表明其可以用于人体，具有安全性，但是没有观察到抗肿瘤效果。
发明内容
为此，本发明所要解决的技术问题在于提供一种基因工程菌VNP20009-M在制备预防和治疗肉瘤药物中的新应用。
为解决上述技术问题，本发明公开了所述的基因工程菌VNP20009-M在制备预防和治疗恶性肉瘤药物中的应用。
进一步的，所述肉瘤为来源于结缔组织和肌肉等间叶组织的恶性肉瘤。
进一步的，所述肉瘤包括发生于脂肪、筋膜、肌肉、纤维、淋巴及血管、骨膜及长骨两端的肉瘤。
进一步的，所述肉瘤包括软组织肉瘤和骨肉瘤。
现有软组织肉瘤患者中，以多形性未分化肉瘤(undifferentiated pleomorphic sarcoma，UPS)最多见，占25-35％；其次是脂肪肉瘤(liposarcoma， LPS)，占25-30％；平滑肌肉瘤(leiomyosarcoma，LMS)12％；滑膜肉瘤(synovial sarcoma，SS)占10％；恶性周围神经鞘膜瘤(malignant peripheral nerve sheath tumor，MPNST)占6％。
骨肉瘤又称为成骨肉瘤，多发生在20岁以下的青少年或儿童。骨肉瘤是从间质细胞系发展而来，由于肿瘤经软骨阶段直接或间接形成肿瘤骨样组织和骨组织使得肿瘤迅速生长。骨肉瘤在小儿骨恶性肿瘤中最为常见，约为小儿肿瘤的5％。
所述肉瘤包括原发肉瘤、术后复发肉瘤、或术后转移至其他部位的肉瘤。
进一步的，所述肉瘤为恶性肉瘤术后转移至肺部的肿瘤。
优选的，所述基因工程菌VNP20009-M的最低有效施用剂量为6.4×10 7CFU/m 2。
上述肿瘤预防和治疗的给药方式可通过多种途径进行给药，包括但不限于：口服给药、局部给药、注射给药(包括但不限于经静脉、腹膜、皮下、肌肉、瘤内给药)等。
如现有技术中已知的，本发明上述基因工程菌VNP20009-M为已知菌株，其性能、形状、构建方法均如中国专利CN105983103A中所记载。
所述基因工程菌VNP20009-M为克隆有L-methioninase基因的减毒鼠伤寒沙门氏菌VNP20009。
进一步的，所述的基因工程菌VNP20009-M为携带了质粒的减毒鼠伤寒沙门氏菌VNP20009，其中，所述质粒上克隆有L-methioninase基因。
所述的基因工程菌VNP20009-M按照如下方法构建得到：将L-methioninase基因亚克隆至质粒中，得到L-methioninase表达质粒，将Lmethioninase表达质粒电转化至减毒鼠伤寒沙门氏菌VNP20009，即得。
所述质粒包括但不限于pSVSPORT质粒、pTrc99A质粒、pcDNA3.1质粒、pBR322质粒或pET23a质粒。
最为优选的是，在构建基因工程菌VNP20009-M的过程中，当选用pSVSPORT质粒时，将L-methioninase基因亚克隆至质粒中，得到L-methioninase表达质粒，然后将Lmethioninase表达质粒电转化至减毒鼠伤寒沙门氏菌VNP20009中得到基因工程菌。
其中，所述的电转化条件为电压2400V，电阻400Ω，电容25μF，放电时间4ms。
本发明还公开了基因工程菌VNP20009-M在制备甲硫氨酸酶剂中的应用。
本发明公开了所述基因工程菌VNP20009-M在现有基础上用于治疗恶性肉瘤的新应用，所述基因工程菌VNP20009-M能够有效杀伤肿瘤细胞，消除肿瘤病灶，对于原发性肉瘤、术后复发肿瘤、以及恶性肉瘤术后转移至其他部位的肿瘤细胞，都具有较好的杀伤效果，治疗效果较佳；尤其是对于软组织肉瘤术后转移至肺部的肿瘤，具有较好的杀伤效果；而且所述基因工程菌对人体无明显毒副作用，为恶性肉瘤的治疗提供了安全有效的新途径。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中，
图1为质粒pSVSPORT-L-methioninase酶切鉴定1％琼脂糖凝胶电泳图；
图2为本发明是Western blot鉴定methioninase表达结果图；
图3为本发明检测沙门氏菌中methioninase活性结果图；
图4为实施例2中患者治疗前的胸部CT检查病灶状况；
图5为实施例2中患者治疗4周后的胸部CT检查结果。
具体实施方式
实施例1 基因工程菌VNP20009-M的构建
本发明所述基因工程菌VNP20009-M的构建方法和过程如中国专利CN105983103A中实施例中所记载。
(1)构建表达L-methioninase基因的质粒。
先合成L-methioninase(GenBank：L43133.1)基因亚克隆至pUC57质粒(金斯瑞公司)，接着通过Kpn I和Hind III酶切位点亚克隆至pSVSPORT质粒(invitrogen)，得到pSVSPORT-L-methioninase表达质粒。具体构建过程如下：
将pSVSPORT质粒用Kpn I和Hind III双酶切，酶切体系为：2μg质粒DNA，3μL 10×buffer，1.5μL Kpn I酶，1.5μL Hind III酶，加入ddH2O补足体积至30μL，37℃温浴3h。然后将酶切体系在1％的琼脂糖凝胶中通过电泳分离，切出4.1kb大小的DNA条带，用胶回收纯化试剂盒纯化DNA。
通过全基因合成得到L-methioninase编码区域DNA片段亚克隆至pUC57质粒(金斯瑞公司)，用Kpn I和Hind III双酶切，酶切体系为：3μg质粒DNA，3μL 10×buffer，1.5μLKpn I酶，1.5μL Hind III酶，加入ddH2O补足体积至30μL，37℃温浴3h。然后将酶切体系在1％的琼脂糖凝胶中通过电泳分离，切出1.2kb大小的DNA条带，用胶回收纯化试剂盒纯化DNA。
将pSVSPORT(Kpn I/Hind III)和L-methioninase编码区域DNA片段(Kpn I/HindIII)连接，连接反应中加入2μL载体、6μL插入片段、1μL T4DNA连接酶，16℃温浴16h。
将连接产物转化到E.coli DH5α(Takara)的感受态细胞中。取一管50μL的DH5α感受态细胞置于冰上，待其融化后，向其中加入5μL上述连接产物，轻弹混匀，后于冰上孵育30min；42℃热击60s，后冰上静置2min；加入500μL无抗性的LB液体培养基，37℃震荡培养1h后涂布在含氨苄青霉素抗性的LB培养基平板上，过夜培养。
克隆生长出来后，挑单克隆菌落至3mL含氨苄青霉素的LB培养液中，37℃震荡培养16h，提取质粒DNA，用Kpn I和Hind III酶切鉴定，阳性克隆中可得到4.1kb、1.2kb的两条DNA条带，如图1所示。再通过测序进一步确定阳性克隆的序列完全正确。
(2)构建携带质粒的VNP20009菌和携带克隆有L-methioninase的基因的质粒的VNP20009菌。
将pSVSPORT和pSVSPORT-L-methioninase表达质粒分别电转化至VNP20009菌株(YS1646，ATCC号202165)，分别命名为VNP20009-V和VNP20009-M。具体构建过程如下：
将感受态细菌VNP20009置于冰上，待其融化后移入预冷电转杯，向其中加入2μL质粒，轻弹混匀，于冰上孵育1min。将电转杯放入电转仪，条件设置为电压2400V，电阻400Ω，电容25μF，放电时间4ms。电击完立刻加入1mL SOC培养基，轻轻混匀。37℃震荡培养1h；用移液器将细菌沉淀吹打均匀后涂布在含氨苄青霉素抗性的LB-O培养基平板上。然后将平板置于37℃温箱培养16h。VNP20009-V和VNP20009-M用LB-O培养后，提取质粒，酶切鉴定正确。
取1×10 8沙门菌用蛋白裂解液提取蛋白，进行10％SDS-PAGE电泳，再稳压冰浴电转至PVDF膜，BSA室温封闭1h后，TBST漂洗3×5min，加入兔抗L-methioninase抗体(1：1000)，4℃孵育过夜。TBST漂洗3次，每次5min再加HRP标记的抗兔二抗(1：10000)，室温孵育1h，TBST漂洗3次，每次5min，ECL化学发光法显影。结果如图2所示，在分子量约43kD处有特异性条带，说明VNP20009-M与VNP20009、VNP20009-V相比，L-methioninase表达量显著提高。
将L-甲硫氨酸和吡哆醛分别与VNP20009-V和VNP20009-M菌体混合，37℃孵育10min后用50％三氯乙酸终止，离心取上清，与3-甲基-2-苯并噻唑啉酮腙盐酸盐水合物(MBTH)充分混匀，50℃孵育30min后，测定320nm处的吸光值，以每分钟催化转化1μmolα-酮丁酸的酶量定义为1个酶活性单 位。结果显示(如图3所示)，沙门菌VNP20009-M的甲硫氨酸酶活性比VNP20009-V高10倍。
可见，所构建基因工程菌沙门菌VNP20009-M具有较高的甲硫氨酸酶活性，可作为甲硫氨酸酶剂的制备之用。
实施例2 基因工程菌VNP20009-M治疗软组织肉瘤的效果
1)过往病史及诊断
临床一男性患者，64岁，左腿肿块在南京市鼓楼医院经穿刺活检鉴定为梭形细胞肉瘤。后行肿瘤切除术，再次取组织分析病理，显示肿瘤细胞SMA(－)、DES(－)、MyoD1(－)、FN(+)、STAT6(－)、CD34(－)、S100(－)、CKpan(－)、EMA(－)、Vimentin(++)，结合HE切片，符合恶性纤维组织细胞瘤。
后定期随访复查CT发现右肺上叶软组织占位，穿刺活检病理显示梭形细胞肉瘤。根据患者既往治疗及相关检查，诊断梭形细胞肉瘤术后复发肺转移。
2)治疗方案
以250ml生理盐水稀释的基因工程菌VNP20009-M通过静脉输入体内，剂量为6.4×10 7CFU/m 2。每次间隔1周，共输液5次。
3)疗效
3.1肿瘤大小变化
治疗前的胸部CT检查显示右肺上叶病灶大小约为18*10*10cm(如图4所示)。治疗4周以后，CT检查显示右肺上叶病灶大小约为17*10*10cm(如图5所示)，与治疗前相比肿瘤大小无明显变化。
3.2肿瘤内部变化
治疗前，CT图像显示肺部病灶内部呈片状液化坏死。如图4所示，白线圈出的病灶内部呈现不均匀的颜色，较黑区域表示此处的细胞已经坏死， 较亮区域表示活细胞。治疗4周后，CT图像显示病灶内部呈均一性结构(如图5所示)，CT值约为10HU。CT值指示物质密度，当它低于20HU表示物质为液态，提示该区域肿瘤细胞基本坏死呈现液态。由此说明，经VNP20009-M治疗后，肉瘤患者的肿瘤细胞快速坏死液化。但是由于病灶在体内，无法抽取液体，所以未能观察到病灶缩小。
3.3副反应
每次治疗当日，输液后5-6小时，患者发热最高39.6度左右，物理降温可恢复正常体温。除此以外，无其他异常不适感觉。治疗期间，检查肝、肾功能等各项指标，结果如下表1所示。检测结果显示，与治疗前相比，患者身体各项指标基本相似。以上结果说明VNP20009-M对患者没有产生额外的毒副作用。
表1 患者身体各项指标数据
项目 参考值 治疗前 治疗1周 治疗4周 治疗8周
丙氨酸氨基转移酶 0--33U/L 20 26 16 19
天冬氨酸氨基转移酶 0--32U/L 16 20 13 21
总胆红素 0--21umol/L 8.3 9.5 11.7 15
碱性磷酸酶 35--104U/L 71 91 96 140
乳酸脱氢酶 109--245U/L 158 160 135 127
白蛋白 35--52g/L 27 30 29 24
尿素氮 2.86--8.21mmol/L 4.36 2.41 3.2 2.23
肌酐 59-104umol/L 55 57 51 52
钾 3.5--5.1mmol/L 3.1 4.14 4.01 3.56
钠 136--145mmol/L 138 134 136 137
血小板 125--35010*9/L 118 153 155 103
以上数据说明，VNP20009-M能够有效杀伤恶性肉瘤细胞，消除肿瘤病灶，而且对人体无严重毒副作用。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保 护范围之中。

Claims (10)

  1. 基因工程菌VNP20009-M在制备预防和治疗恶性肉瘤药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述肉瘤为来源于结缔组织和肌肉等间叶组织的恶性肉瘤。
  3. 根据权利要求2所述的应用，其特征在于，所述肉瘤包括发生于脂肪、筋膜、肌肉、纤维、淋巴及血管、骨膜及长骨两端的肉瘤。
  4. 根据权利要求3所述的应用，其特征在于，所述肉瘤包括软组织肉瘤和骨肉瘤。
  5. 根据权利要求1-4任一项所述的应用，其特征在于，所述肉瘤包括原发肉瘤、术后复发肉瘤、或术后转移至其他部位的肉瘤。
  6. 根据权利要求5所述的应用，其特征在于，所述肉瘤为恶性肉瘤术后转移至肺部的肿瘤。
  7. 根据权利要求1-6任一项所述的应用，其特征在于，所述基因工程菌VNP20009-M的最低有效施用剂量为6.4×10 7CFU/m 2。
  8. 根据权利要求1-7任一项所述的应用，其特征在于，所述基因工程菌VNP20009-M为克隆有L-methioninase基因的减毒鼠伤寒沙门氏菌VNP20009。
  9. 根据权利要求8所述的应用，其特征在于，所述的基因工程菌VNP20009-M为携带了质粒的减毒鼠伤寒沙门氏菌VNP20009，所述质粒上克隆有L-methioninase基因。
  10. 根据权利要求8或9任一项所述的应用，其特征在于，所述的基因工程菌VNP20009-M按照如下方法构建得到：将L-methioninase基因亚克隆至质粒中，得到L-methioninase表达质粒，将Lmethioninase表达质粒电转化至减毒鼠伤寒沙门氏菌VNP20009，即得。
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AU2018241657B2 (en) 2020-11-05
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EP3583956A4 (en) 2020-02-12
US11324781B2 (en) 2022-05-10
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