肺癌靶向治疗的疱疹溶瘤病毒开发研究

Oncolytic virotherapy exploits the ability of viruses to infect and kill cancer cells. Numerous clinical trials demonstrated that oncolytic herpes simplex virus 1 (oHSV) is effective against a subpopulation of solid cancers, and can be administered repeatedly. Yet it is confronted with problems as to treatment of lung cancer for minor entry of viruses may cause "cytokine storm" to lung and functionally destroy it. Building on my long-term experience on the molecular mechanisms by which HSV-1 enters cells, I have devised a strategy to re-design HSVs, such that they exclusively infect lung cancer cells that carry on their surface a specific receptor of choice (retargeting). They no longer infect non-cancer cells. Once the retargeted HSVs enter the specific lung cancer cells, they counteract innate responses and destroy the cancer cells. The aims of this proposal are: AIM 1, replace the Ig-folded core in the receptor-binding virion glycoprotein gD with three anti-lung cancer surface protein single-chain antibodies. AIMs 2: design robust retargeted-HSVs based on wide-type virus to boost its anti-cancer capability. AIM 3: express SOCS3 to inhibit "cytokine storm" and delete "danger antigens" from HSV-1 viral proteins to avoid potentially fatal immune reaction. AIMs 4: develop cancer cell lines and animal models acceptable for evaluation of retargeted-HSVs.

减毒性疱疹溶瘤病毒(oHSV)在国外进入不同的实体瘤治疗临床试验，但在肺癌治疗中遇到障碍。原因是微量病毒进入肺细胞，可能激起免疫应激反应导致肺功能的丧失，针对肺癌治疗设计靶向性疱疹溶瘤病毒迫在眉睫。申请人是靶向性疱疹溶瘤病毒的发明人，研究获4项国际专利，成果被应用在恶性脑瘤，乳腺癌和卵巢癌的治疗中。本研究设计针对肺癌细胞表面蛋白，构建靶向性疱疹溶瘤病毒oHSV，达到靶向高效安全的治疗目的。靶向性方面，将三种不同类型的肺癌表面蛋白的单链抗体（ScFv）在病毒受体识别蛋白gD中定点表达，再敲除gD对正常细胞的识别，使病毒只识别肺癌细胞；高效性方面，以野生型病毒为模板，保持天然活力；安全性方面，在病毒中表达抑制"细胞因子风暴"的SOCS3；同时敲除病毒"危险抗原" 基因，弱化免疫应激反应。构建的重组病毒将在相应的肺癌细胞表面蛋白细胞系和动物模型中进行鉴定评估，为新药开发提供安全高效的保障。

本研究设计针对肺癌构建了靶向性疱疹溶瘤病毒oHSV，在溶瘤病毒中表达外源性的细胞因子IL2，使得溶瘤病毒在肿瘤细胞中复制溶瘤地同时，在肿瘤微环境中表达细胞因子，通过最终促进肿瘤微环境中的IFN-γ来达到摧毁癌症的效果，具有副作用小，效果特异的作用。用它在体外进行了两种肺癌细胞的共培养实验，结果 ，0.1-1PFU/cell的病毒，足以在48小时摧毁50-60%的细胞，96小时细胞只剩下将10%左右，而对照组的细胞存活率是100%；在小鼠体内研究中完成了小鼠成瘤和溶瘤病毒的单次注射、三次注射抗瘤试验，在三次注射的小鼠肺癌模型中显示90%以上的抑瘤率；此外，完成了重组病毒表达SOCS4 的病毒构建，成功表达外源SOCS4，通过小鼠的病毒气管注射，通过检测MCP-1, IL-1β, TNF-α, IL-6, 和 IFN-γ, 比较它与野生病毒在血液和肺器官中的刺激产生的浓度，证明携带SOCS4的病毒的确能够抑制“细胞因子风暴”，为溶瘤病毒在肺癌中的应用治疗提供理论基础。