WO2018201602A1 - 重组溶瘤ii型单纯疱疹病毒在制备抗淋巴瘤、食道癌、乳腺癌、胰腺癌药物中的应用 - Google Patents

重组溶瘤ii型单纯疱疹病毒在制备抗淋巴瘤、食道癌、乳腺癌、胰腺癌药物中的应用 Download PDF

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WO2018201602A1
WO2018201602A1 PCT/CN2017/091625 CN2017091625W WO2018201602A1 WO 2018201602 A1 WO2018201602 A1 WO 2018201602A1 CN 2017091625 W CN2017091625 W CN 2017091625W WO 2018201602 A1 WO2018201602 A1 WO 2018201602A1
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herpes simplex
virus
simplex virus
recombinant oncolytic
tumor
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PCT/CN2017/091625
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刘滨磊
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武汉滨会生物科技股份有限公司
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Priority to US16/671,205 priority Critical patent/US20200061133A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16621—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16632—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

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  • the invention relates to the field of biomedicine, in particular to the application of recombinant oncolytic type II herpes simplex virus in preparing medicaments for anti-lymphoma, esophageal cancer, breast cancer and pancreatic cancer.
  • Herps simplex virus is a double-stranded DNA virus of about 154 kb long that replicates in the nucleus of infected host cells.
  • HSV vectors have the following advantages: 1) a wide range of host cells; 2) high viral titers; 3) large foreign gene capacity.
  • a disadvantage of the HSV vector is its toxicity.
  • the prior art discloses a patent document "a recombinant oncolytic type II herpes simplex virus, a preparation method and application thereof, and a tumor diagnosis kit", which comprises a herpes simplex virus type II, and the Latin literature name is Herpes Simplex Virus Type 2 On February 3, 2010, it was deposited with the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee, No. 3, Beichen West Road, Chaoyang District, Beijing, with the preservation number CGMCC No. 3600.
  • CGMCC General Microbiology Center
  • H2d3d4-hGF strain has the meaning of strain: H2 refers to type II herpes simplex virus HG52 strain (oHSV2); d3 refers to ICP34.5; d4 refers to ICP47; hGF refers to insertion of human granulocyte-macro Phage colony stimulating factor (hGM-CSF) expression cassette.
  • oHSV2 type II herpes simplex virus HG52 strain
  • d3 refers to ICP34.5
  • d4 refers to ICP47
  • hGF refers to insertion of human granulocyte-macro Phage colony stimulating factor (hGM-CSF) expression cassette.
  • hGM-CSF human granulocyte-macro Phage colony stimulating factor
  • the invention is based on the continuous research on the above virus, and finds a new use of the virus, and provides the application of the recombinant oncolytic type II herpes simplex virus in preparing anti-lymphoma, esophageal cancer, breast cancer and pancreatic cancer drugs.
  • the recombinant oncolytic type II herpes simplex virus provided by the present invention is a recombinant oncolytic type II herpes simplex virus (oHSV2) which excludes the ICP34.5 and ICP47 genes of the wild type II herpes simplex virus HG52 strain.
  • the virus strain has been deposited with the Common Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee as described in the background art, and the preservation number is CGMCC No. 3600.
  • CGMCC Common Microbiology Center
  • the invention has confirmed by research and experiment that the oHSV2 virus strain has outstanding oncolytic effect against lymphoma, esophageal cancer, breast cancer and pancreatic cancer, indicating that the oHSV2 virus or oHSV2 viral vector has anti-lymphoma, esophageal cancer and breast cancer preparation.
  • pancreatic cancer drugs The application prospects of pancreatic cancer drugs.
  • the invention has the beneficial effects of discovering the new biological function of the existing recombinant oncolytic type II herpes simplex virus, which can prepare medicine for treating anti-lymphoma, esophageal cancer, breast cancer and pancreatic cancer, and the recombinant oncolytic type II herpes simplex
  • the virus can specifically select to grow and multiply in human tumor cells, does not affect normal cell proliferation, and thus can effectively kill cancer cells, and the effect is good.
  • Figure 1 is a graph showing changes in tumor diameter of right tumor after injection of oHSV2 in tumor-bearing mice of the A20 mouse lymphoma model.
  • Figure 2 is a graph showing changes in tumor diameter of left tumor after injection of oHSV2 in tumor-bearing mice of the A20 mouse lymphoma model.
  • Figure 3 is a graph showing changes in tumor diameter after injection of oHSV2 in tumor-bearing mice of the 4T1 mouse breast cancer model.
  • Figure 4 is a graph showing changes in tumor diameter after injection of oHSV2 in nude mice bearing MDA-MB-231 human breast cancer model.
  • Figure 5 is a comparison of tumor diameters after injection of oHSV2 in tumor-bearing SCID mice of the EC109 esophageal cancer model.
  • Figure 6 is a graph showing changes in tumor diameter after injection of oHSV2 in tumor-bearing nude mice of the MiaPaCa-2 human pancreatic cancer model.
  • Figure 7 is a graph showing changes in tumor diameter after injection of oHSV2 in tumor-bearing mice of the B16 mouse melanoma model.
  • Figure 8 is a graph showing changes in tumor diameter after injection of oHSV2 in tumor-bearing nude mice of the SNK-6 human NKT lymphoma model.
  • Figure 9 is a graph showing the comparison of virus titers after oHSV2 infection of four tumor cells.
  • A20 mouse B cell lymphoma cells were injected subcutaneously into bilateral flank, at a dose of 2 ⁇ 10 6 , and 20 female BALB/c mice (10/group) were induced to produce tumors.
  • the schedule of three injections of virus in the right flank tumor is shown in the table below.
  • Fig. 1 shows that after three injections of the virus in the right tumor, the tumor in the treatment side virus treatment group was significantly smaller than that in the control group.
  • the mean tumor diameters of the control group and the virus treatment group were 1.59 and 0 cm, respectively.
  • the diameter of the tumor in the drug-administered group was significantly different from that in the control group (p ⁇ 0.01).
  • Figure 2 shows that the virus also has a good anti-tumor effect on the non-treated side (left side) tumor.
  • the average tumor diameter of the control group and the virus treatment group was 1.54 and 0.4 cm, respectively, 28 days after the first injection of the virus.
  • the diameter of the left tumor in the drug-administered group was significantly different from that in the control group (p ⁇ 0.05).
  • Example 2 Anticancer effect of recombinant oncolytic type II herpes simplex virus against breast cancer
  • 4T1 mouse breast cancer cells were subcutaneously injected into the right flank at a dose of 1 ⁇ 10 6 , and 20 female BALB/c mice (10/group) were induced to produce tumors. The timing of three injections of virus in the tumor is shown in the table below.
  • the tumors in the virus-treated group were significantly smaller than those in the control group.
  • the mean tumor diameters of the control group and the virus treatment group were 1.83 and 0.87 cm, respectively.
  • the diameter of the tumor in the drug-administered group was significantly different from that in the control group (p ⁇ 0.05). The experimental results are shown in Figure 3.
  • MDA-MB-231 human breast cancer cells were injected subcutaneously into the right flank at a dose of 1 ⁇ 10 6 to induce tumor formation in 20 female BALB/c nude mice (10/group). The timing of three injections of virus in the tumor is shown in the table below.
  • the tumors in the virus-treated group were significantly smaller than those in the control group.
  • the mean tumor diameters of the control group and the virus treatment group were 1.24 and 0.21 cm, respectively.
  • the diameter of the tumor in the drug-administered group was significantly different from that in the control group (p ⁇ 0.01). The experimental results are shown in Figure 4.
  • Example 3 Anticancer effect of recombinant oncolytic type II herpes simplex virus (oHSV2) against esophageal cancer
  • oHSV2 herpes simplex virus
  • EC109 human esophageal cancer cells were injected subcutaneously into the right flank, at a dose of 1 ⁇ 10 6 , and 20 female SCID immunodeficiency mice (10/group) were induced to produce tumors.
  • the schedule of three injections of virus in the tumor is shown in the table below.
  • the tumors in the virus-treated group were significantly smaller than those in the control group.
  • the mean tumor diameters of the control group and the virus treatment group were 1.64 and 0.14 cm, respectively.
  • the diameter of the tumor in the drug-administered group was significantly different from that in the control group (p ⁇ 0.01). The experimental results are shown in Figure 5.
  • MiaPaCa-2 human pancreatic cancer cells were injected subcutaneously into the right flank at a dose of 1 ⁇ 10 6 to induce tumor formation in 20 female nude mice (10/group). The schedule of three injections of virus in the tumor is shown in the table below.
  • the tumors in the virus-treated group were significantly smaller than those in the control group.
  • the average tumor diameter of the control group and the virus treatment group was 1.45 and 0.47 cm, respectively, 28 days after the first injection of the virus.
  • the diameter of the tumor in the drug-administered group was significantly different from that in the control group (p ⁇ 0.05).
  • the experimental results are shown in Figure 6.
  • B16 melanoma cells were injected subcutaneously into the right flank at a dose of 2 ⁇ 10 5 to induce tumor formation in 20 female C57/BL mice (10/group).
  • the schedule of three injections of virus in the tumor is shown in the table below.
  • the tumors in the virus-treated group did not significantly shrink compared with the control group.
  • the mean tumor diameters of the control group and the virus treatment group were 1.44 and 1.50 cm, respectively.
  • the diameter of the tumor in the drug-administered group was not significantly different from that in the control group.
  • the experimental results are shown in Figure 7.
  • Recombinant oncolytic type II herpes simplex virus (oHSV2) has no significant anti-tumor effect on murine B16 melanoma (B16).
  • SNK-6 human NKT lymphoma cells were injected subcutaneously into the right flank at a dose of 2 ⁇ 10 6 to induce tumor formation in 20 female BALB/c nude mice (10/group). The schedule of three injections of virus in the tumor is shown in the table below.
  • the tumors in the virus-treated group did not significantly shrink compared with the control group.
  • the mean tumor diameters of the control group and the virus treatment group were 1.80 and 1.72 cm, respectively.
  • the diameter of the tumor in the drug-administered group was not significantly different from that in the control group.
  • the experimental results shown in Figure 8 showed that recombinant oncolytic type II herpes simplex virus (oHSV2) had no significant inhibitory effect on human NKT lymphoma (SNK-6).
  • the cells used in the experiment were as follows: four kinds of Vero cells: African green monkey kidney cells, B16 cells: murine melanoma cells, SNK-6 cells: human NKT lymphoma cells, CT26 cells: murine colon cancer cells
  • the virus was significantly increased in Vero cells, and the virus titer began to rise rapidly at 8 hours after infection, slowly rising after 24 hours, and peaked at 48 hours (lg6.3 CCID50/ml).
  • the virus has a certain value in CT-26, and the titer reaches lg 3.97CCID50/ml after 24 hours and is maintained for 72 hours.
  • the virus does not add value in B16 and SNK-6 cells.
  • Vero cells African green monkey kidney cells, herpes simplex virus (HSV) sensitive cells, HSV infected Vero cells can achieve higher titers after breeding.
  • HSV herpes simplex virus
  • B16 cells murine melanoma cells, HSV non-sensitive cells, do not support HSV reproduction, resulting in oHSV2 can not kill B16 cells.
  • SNK-6 cells human NKT lymphoma cells, HSV non-sensitive cells, do not support HSV reproduction, resulting in oHSV2 can not kill SNK-6 cells.
  • CT26 cells murine colon cancer cells, HSV semi-sensitive cells, HSV can be propagated in this cell, but the titer is low.
  • oHSV2 virus cannot proliferate in B16 and SNK-6 cells is that both cells lack viral receptors. Therefore, recombinant oncolytic type II herpes simplex virus (oHSV2) does not have an oncolytic effect on all tumors.

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Abstract

一种重组溶瘤II型单纯疱疹病毒在制备抗淋巴瘤、食道癌、乳腺癌、胰腺癌药物中的应用。该重组溶瘤II型单纯疱疹病毒能特异性选择在人肿瘤细胞内生长繁殖，不影响正常细胞增殖。

Description

重组溶瘤II型单纯疱疹病毒在制备抗淋巴瘤、食道癌、乳腺癌、胰腺癌药物中的应用 技术领域
本发明涉及生物医药领域，具体地指重组溶瘤II型单纯疱疹病毒在制备抗淋巴瘤、食道癌、乳腺癌、胰腺癌药物中的应用。
背景技术
单纯疱疹病毒(Herps simplex virus，HSV)是一种长约154kb的双链DNA病毒，可在受染宿主细胞核中复制。HSV载体具有以下优点：1)宿主细胞广泛；2)病毒滴度高；3)外源基因容量大。HSV载体的缺点在于它的毒性。
现有技术公开了“一种重组溶瘤II型单纯疱疹病毒、其制备方法及应用和肿瘤诊断试剂盒”的专利文件，其中包含一个II型单纯疱疹病毒，拉丁文学名为Herpes Simplex Virus Type 2，于2010年2月03日保藏至位于北京市朝阳区北辰西路1号院3号的中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC No.3600。被保藏的生物材料H2d3d4-hGF株，其株号的含义为：H2指II型单纯疱疹病毒HG52株(oHSV2)；d3指剔除ICP34.5；d4指剔除ICP47；hGF指插入人粒细胞-巨噬细胞集落刺激因子(hGM-CSF)表达盒。该专利文件中公开了此重组溶瘤II型单纯疱疹病毒能够用于多种肿瘤的体内溶瘤。
发明内容
本发明基于对上述病毒的不断研究，发现了该病毒新的用途，提供了该重组溶瘤II型单纯疱疹病毒在制备抗淋巴瘤、食道癌、乳腺癌、胰腺癌药物中的应用。
本发明提供的重组溶瘤II型单纯疱疹病毒，是剔除了野生II型单纯疱疹病毒HG52株的ICP34.5及ICP47基因的重组溶瘤II型单纯疱疹病毒(oHSV2)。该病毒株已如背景技术所述保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC No.3600。
本发明中的重组溶瘤II型单纯疱疹病毒(oHSV2)的制备过程及其可能的药物剂型已经在中国发明专利“重组Ⅱ型单纯疱疹病毒载体及其制备方法、重组病毒、药物组合物及应用”中公开(授权公告号CN 102146418B)，此处不做赘述。
本发明通过研究及实验证实，oHSV2病毒株针对淋巴瘤、食道癌、乳腺癌、胰腺癌具有突出的溶瘤抑瘤效果，说明oHSV2病毒或oHSV2病毒载体具有制备抗淋巴瘤、食道癌、乳腺癌、胰腺癌药物的应用前景。
本发明的有益效果：发现了现有重组溶瘤II型单纯疱疹病毒新的生物学功能，它能够制备治疗抗淋巴瘤、食道癌、乳腺癌、胰腺癌药物，该重组溶瘤II型单纯疱疹病毒能特异性选择在人肿瘤细胞内生长繁殖，不影响正常细胞增殖，从而能够有效杀伤癌细胞，效果好，这些新的适应症，为该重组溶瘤II型单纯疱疹病毒药物的开发利用提供了更多依据。
附图说明
图1为A20小鼠淋巴瘤模型的荷瘤小鼠注射oHSV2后右侧瘤的瘤体直径变化图。
图2为A20小鼠淋巴瘤模型的荷瘤小鼠注射oHSV2后左侧瘤的瘤体直径变化图。
图3为4T1小鼠乳腺癌模型的荷瘤小鼠注射oHSV2后瘤体直径变化图。
图4为MDA-MB-231人乳腺癌模型的荷瘤裸鼠注射oHSV2后瘤体直径变化图。
图5为EC109食道癌模型的荷瘤SCID鼠注射oHSV2后瘤体直径对比图。
图6为MiaPaCa-2人胰腺癌模型的荷瘤裸鼠注射oHSV2后瘤体直径变化图。
图7为B16小鼠黑色素瘤模型的荷瘤小鼠注射oHSV2后瘤体直径变化图。
图8为SNK-6人NKT淋巴瘤模型的荷瘤裸鼠注射oHSV2后瘤体直径变化图。
图9为oHSV2感染四种肿瘤细胞后的病毒滴度对比图。
具体实施方式
以下结合具体实施例对本发明作进一步详细描述。下述实施例中所用方法如无特别说明均为常规方法，所有引物合成及测序工作均由上海生工完成。
实施例1重组溶瘤II型单纯疱疹病毒(oHSV2)抗淋巴瘤的抑瘤效果
于双侧胁腹皮下注射A20小鼠B细胞淋巴瘤细胞，剂量为2×106，诱导20只雌性BALB/c小鼠(10只/组)产生肿瘤。于右侧胁腹瘤内三次注射病毒的时间安排见下表。
表1.给A20荷瘤小鼠注射病毒的时间安排
Figure PCTCN2017091625-appb-000001
结果，图1显示右侧瘤内三次注射病毒后，治疗侧病毒治疗组的肿瘤与对照组肿瘤相比，显著缩小。首次注射病毒后28天，对照组、病毒治疗组的平均肿瘤直径分别为1.59和0cm。给药组肿瘤的直径与对照组相比有显著差异(p<0.01)。
同样地，图2显示病毒对于非治疗侧(左侧)肿瘤也有较好的抗肿瘤作用，首次注射病毒后28天，对照组、病毒治疗组的平均肿瘤直径分别为1.54和0.4cm。给药组左侧肿瘤直径与对照组相比有显著差异(p<0.05)。
实施例2重组溶瘤II型单纯疱疹病毒抗乳腺癌的抗癌效果
(1)重组溶瘤II型单纯疱疹病毒(oHSV2)抗鼠乳腺癌(4T1)的抑瘤效果
于右侧胁腹皮下注射4T1小鼠乳腺癌细胞，剂量为1×106，诱导20只雌性BALB/c小鼠(10只/组)产生肿瘤。瘤内三次注射病毒的 时间安排见下表。
表2.给4T1荷瘤小鼠注射病毒的时间安排
Figure PCTCN2017091625-appb-000002
瘤内三次注射病毒后，病毒治疗组的肿瘤与对照组肿瘤相比，显著缩小。首次注射病毒后28天，对照组、病毒治疗组的平均肿瘤直径分别为1.83和0.87cm。给药组肿瘤的直径与对照组相比有显著差异(p<0.05)。实验结果如图3所示。
(2)重组溶瘤II型单纯疱疹病毒(oHSV2)抗人乳腺癌(MDA-MB-231)的抑瘤效果
于右侧胁腹皮下注射MDA-MB-231人乳腺癌细胞，剂量为1×106，诱导20只雌性BALB/c裸鼠(10只/组)产生肿瘤。瘤内三次注射病毒的时间安排见下表。
表3.给MDA-MB-231荷瘤裸鼠注射病毒的时间安排
Figure PCTCN2017091625-appb-000003
瘤内三次注射病毒后，病毒治疗组的肿瘤与对照组肿瘤相比，显著缩小。首次注射病毒后28天，对照组、病毒治疗组的平均肿瘤直径分别为1.24和0.21cm。给药组肿瘤的直径与对照组相比有显著差异(p<0.01)。实验结果如图4所示。
实施例3重组溶瘤II型单纯疱疹病毒(oHSV2)抗食道癌的抗癌效果
于右侧胁腹皮下注射EC109人食道癌细胞，剂量为1×106，诱导20只雌性SCID免疫力缺陷鼠(10只/组)产生肿瘤。于瘤内三次注射病毒的时间安排见下表。
表4.给EC109荷瘤SCID鼠注射病毒的时间安排
Figure PCTCN2017091625-appb-000004
瘤内三次注射病毒后，病毒治疗组的肿瘤与对照组肿瘤相比，显著缩小。首次注射病毒后28天，对照组、病毒治疗组的平均肿瘤直径分别为1.64和0.14cm。给药组肿瘤的直径与对照组相比有显著差异(p<0.01)。实验结果如图5所示。
实施例4重组溶瘤II型单纯疱疹病毒(oHSV2)抗胰腺癌的抗癌效果
于右侧胁腹皮下注射MiaPaCa-2人胰腺癌细胞，剂量为1×106，诱导20只雌性裸鼠(10只/组)产生肿瘤。于瘤内三次注射病毒的时间安排见下表。
表5.给MiaPaCa-2荷瘤裸鼠注射病毒的时间安排
Figure PCTCN2017091625-appb-000005
Figure PCTCN2017091625-appb-000006
瘤内三次注射病毒后，病毒治疗组的肿瘤与对照组肿瘤相比，显著缩小。首次注射病毒后28天，对照组、病毒治疗组的平均肿瘤直径分别为1.45和0.47cm。给药组肿瘤的直径与对照组相比有显著差异(p<0.05)。实验结果如图6所示。
对照例1重组溶瘤II型单纯疱疹病毒(oHSV2)对鼠B16黑色素瘤(B16)的抑瘤效果
于右侧胁腹皮下注射B16黑色素瘤细胞，剂量为2×105，诱导20只雌性C57/BL小鼠(10只/组)产生肿瘤。于瘤内三次注射病毒的时间安排见下表。
表6.给B16荷瘤小鼠注射病毒的时间安排
Figure PCTCN2017091625-appb-000007
瘤内三次注射病毒后，病毒治疗组的肿瘤与对照组肿瘤相比，无明显缩小。首次注射病毒后28天，对照组、病毒治疗组的平均肿瘤直径分别为1.44和1.50cm。给药组肿瘤的直径与对照组相比无显著差异。实验结果如图7所示，重组溶瘤II型单纯疱疹病毒(oHSV2)对鼠B16黑色素瘤(B16)的无明显抑瘤效果。
对照例2重组溶瘤II型单纯疱疹病毒(oHSV2)对人NKT淋巴瘤(SNK-6)的抑瘤效果
于右侧胁腹皮下注射SNK-6人NKT淋巴瘤细胞，剂量为2×106，诱导20只雌性BALB/c裸鼠(10只/组)产生肿瘤。于瘤内三次注射病毒的时间安排见下表。
表7.给SNK-6荷瘤裸鼠注射病毒的时间安排
Figure PCTCN2017091625-appb-000008
瘤内三次注射病毒后，病毒治疗组的肿瘤与对照组肿瘤相比，无明显缩小。首次注射病毒后28天，对照组、病毒治疗组的平均肿瘤直径分别为1.80和1.72cm。给药组肿瘤的直径与对照组相比无显著差异。实验结果如图8所示重组溶瘤II型单纯疱疹病毒(oHSV2)对人NKT淋巴瘤(SNK-6)的无明显抑瘤效果。
实验例1重组溶瘤II型单纯疱疹病毒(oHSV2)在4种细胞系中生长曲线
实验用到的细胞有如下四种Vero细胞：非洲绿猴肾细胞、B16细胞：鼠黑色素瘤细胞、SNK-6细胞：人NKT淋巴瘤细胞、CT26细胞：鼠结肠癌细胞
实验步骤：将4种细胞铺培养板，当细胞汇合度达90％或以上时，按MOI＝0.01用oHSV2病毒感染细胞,在0、4、8、16、24、48及 72小时时间点检测病毒滴度。
结果如图9所示，病毒在Vero细胞中增值明显，病毒滴度在感染后8小时开始迅速升高，24小时后缓慢上升，48小时达到高峰(lg6.3CCID50/ml)。病毒在CT-26中有一定增值，24小时后滴度达lg 3.97CCID50/ml并维持到72小时。病毒在B16和SNK-6细胞中不增值。
结果分析如下：
Vero细胞：非洲绿猴肾细胞，单纯疱疹病毒(HSV)敏感细胞，HSV感染Vero细胞后繁殖可达到较高的滴度。
B16细胞：鼠黑色素瘤细胞，HSV非敏感细胞，不支持HSV繁殖，导致oHSV2不能杀伤B16细胞。
SNK-6细胞：人NKT淋巴瘤细胞，HSV非敏感细胞，不支持HSV繁殖，导致oHSV2不能杀伤SNK-6细胞。
CT26细胞：鼠结肠癌细胞，HSV半敏感细胞，HSV可在此细胞中繁殖，但滴度较低。
结论：oHSV2病毒在B16和SNK-6细胞中不能增殖的原因是两种细胞缺乏病毒受体。因此，重组溶瘤II型单纯疱疹病毒(oHSV2)并非对所有的肿瘤都具有溶瘤抑瘤效果。

Claims (4)

  1. 一种重组溶瘤II型单纯疱疹病毒在制备抗淋巴瘤药物中的应用，其特征在于：所述重组溶瘤II型单纯疱疹病毒为剔除了野生II型单纯疱疹病毒HG52株的ICP34.5及ICP47基因的重组溶瘤II型单纯疱疹病毒。
  2. 一种重组溶瘤II型单纯疱疹病毒在制备抗食道癌药物中的应用，其特征在于：所述重组溶瘤II型单纯疱疹病毒为剔除了野生II型单纯疱疹病毒HG52株的ICP34.5及ICP47基因的重组溶瘤II型单纯疱疹病毒。
  3. 一种重组溶瘤II型单纯疱疹病毒在制备抗乳腺癌药物中的应用，其特征在于：所述重组溶瘤II型单纯疱疹病毒为剔除了野生II型单纯疱疹病毒HG52株的ICP34.5及ICP47基因的重组溶瘤II型单纯疱疹病毒。
  4. 一种重组溶瘤II型单纯疱疹病毒在制备抗胰腺癌药物中的应用，其特征在于：所述重组溶瘤II型单纯疱疹病毒为剔除了野生II型单纯疱疹病毒HG52株的ICP34.5及ICP47基因的重组溶瘤II型单纯疱疹病毒。
PCT/CN2017/091625 2017-05-03 2017-07-04 重组溶瘤ii型单纯疱疹病毒在制备抗淋巴瘤、食道癌、乳腺癌、胰腺癌药物中的应用 WO2018201602A1 (zh)

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