Cloning of carrier cells infected with oncolytic adenovirus driven by midkine promoter and biosafety studies
Cloning of carrier cells infected with oncolytic adenovirus driven by midkine promoter and biosafety studies
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DOI：
10.1002/jgm.3064
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发表时间：
2019-02-01
影响因子：
3.5
通讯作者：
Sugiyama，Takashi
中科院分区：
文献类型：
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作者：
Hamada，Katsuyuki;Takagi，Soichi;Sugiyama，Takashi
A549携带细胞感染溶瘤腺病毒可诱导皮下卵巢肿瘤的完全减瘤，但不能诱导腹膜内播散性卵巢肿瘤的减瘤。这似乎是由于A549载体细胞的抗肿瘤作用不足所致。因此，在本研究中，我们克隆了一种新型的载体细胞，旨在提高其抗肿瘤效果。方法用有限稀释法克隆携带amidkine型启动子的溶瘤腺病毒AdE3-Midkine的载体细胞。我们在同基因小鼠模型中检测了这些细胞对皮下和腹膜内OVHM卵巢肿瘤的抗肿瘤作用。结果克隆了EHMK-51-35载体细胞，其体外抗肿瘤活性是A549载体细胞的10倍。EHMK-51-35载体细胞联合感染AdE3-Midkine和Ad-MGM-CSF后，皮下肿瘤完全减少率为100%，腹膜内播散性肿瘤减少率为60%。用EHMK-51-35载体细胞联合感染AdE3-Midkine和Ad-CGM-CSF对Beagle犬进行单次急性毒性试验，未见严重毒副作用。在血液、唾液、粪便、尿液或整个器官中未检测到具有生物活性的腺病毒。在慢性毒性实验中，用携带AdE3-Midkine基因的EHMK-51-35载体细胞对兔VX2肿瘤进行了5次注射，未见严重毒副作用。结论克隆的EHMK-51-35载体细胞对卵巢肿瘤有显著的抗肿瘤作用，毒性实验证实其安全性。这些发现将扩展到使用狗和猫进行临床前疗效研究，目的是对难治性实体肿瘤进行人类临床试验。
BackgroundA549 carrier cells infected with oncolytic adenovirus can induce complete tumor reduction of subcutaneous ovarian tumors but not intraperitoneal disseminated ovarian tumors. This appears to be a result of the insufficient antitumor effect of A549 carrier cells. Therefore, in the present study, we cloned a novel carrier cell with the aim of improving the antitumor effects.MethodsCarrier cells infected with oncolytic adenovirus AdE3‐midkinewith amidkinepromoter were cloned by limiting dilution. We examined the antitumor effects of these cells on subcutaneous and intraperitoneal OVHM ovarian tumors in a syngeneic mouse model. Biosafety tests were conducted in beagle dogs and rabbits.ResultsWe cloned EHMK‐51‐35 carrier cells with 10‐fold higher antitumor effects compared to A549 carrier cellsin vitro. EHMK‐51‐35 carrier cells co‐infected with AdE3‐midkineand Ad‐mGM‐CSFinduced a 100% complete tumor reduction in subcutaneous tumors and a 60% reduction of intraperitoneal disseminated tumors. Single‐dose acute toxicity test on beagle dogs with EHMK‐51‐35 carrier cells co‐infected with AdE3‐midkineand Ad‐cGM‐CSFshowed no serious side effects. Biologically active adenoviruses were not detected in the blood, saliva, feces, urine or whole organs. In a chronic toxicity test, VX2 tumors in rabbits were injected five times with EHMK‐51‐35 carrier cells infected with AdE3‐midkineand these rabbits showed no serious side effects.ConclusionsSignificant antitumor effects and safety of cloned EHMK‐51‐35 carrier cells were confirmed in intraperitoneal ovarian tumors and toxicity tests, respectively. These findings will be extended to preclinical efficacy studies using dogs and cats, with the aim of conducting human clinical trials on refractory solid tumors.