用B型腺病毒Ad11突变体治疗转移和扩散肿瘤

Oncolytic adenoviruses based on serotype 5 (Ad5) have been developed as a novel treatment strategy for cancer. However, there are several shortcomings with Ad5, including the downregulation of its receptor, the Coxsackie and adenovirus receptor (CAR) in cancer cells, a high prevalence of neutralizing antibodies and hepatotoxicity. Another adenoviral serotype, Ad11, could overcome these obstacles. Our preliminary data in support of this proposal demonstrated that human cancer cell lines expressed significantly higher levels of the Ad11 receptor CD46, resulting in much higher infectivity than Ad5. Stepwise investigations revealed that it was the transcription of Ad11 E1A, not cellular CD46 expression nor virus infectivity, that determined the cell's sensitivity to Ad11 killing. We recently have created a Ad11 mutant (Ad11-Ad5-EP) in which the enhancer-promoter was replaced by that of Ad5. Ad11-Ad5-EP demonstrated increased E1A mRNA levels and replication, together with enhanced oncolytic potency in vitro and in vivo. This effect was found not only in the Ad5-sensitive but also in the Ad11-sensitive cancer cells, broadening the range of tumors that could be effectively killed by Ad11-Ad5-EP. As such, Ad11-Ad5-EP provides a novel backbone for the development of safe, efficient and tumor-selective oncolytic viruses. In this project, we will make a new replication-selective oncolytic adenovirus based on this novel mutant of Ad11-Ad5-EP by manipulation of the E1A, E1B 21K and E3 18.5K genes of Ad11, and evaluate its antitumour efficacy and safety in vitro and in vivo, especially the potential for systemic treatment of metastatic and peritoneally spreading tumors. This new replication-selective oncolytic adenovirus will provide a new therapeutic agent for treatment of most of human cancers.

以Ad5为基础的溶瘤腺病毒已经发展为一种新的肿瘤治疗手段。但Ad5具有几个显著的缺陷：多数肿瘤细胞中受体表达下调；高度普遍的中和抗体和肝毒性。B亚群腺病毒11能克服这些缺陷。我们前期的结果证实人肿瘤细胞显著地高表达Ad11受体CD46，对Ad11感染性更高；但Ad11对肿瘤细胞的杀伤能力取决于早期基因E1 A的转录水平。因此，我们构建了具有Ad5 E1 A增强子和启动子的Ad11-Ad5-EP，发现其在Ad5和Ad11敏感的肿瘤细胞中E1 A的转录和复制均增强，在体内、外有更强的溶瘤能力。因而，Ad11-Ad5-EP为发展安全、高效和肿瘤选择性的溶瘤病毒提供了新的病毒骨架。在本研究中，我们将以新型的突变体Ad11-Ad5-EP为基础，删除病毒三个内源基因构建新型的复制选择性溶瘤腺病毒，并在体内、外评估其抗肿瘤效果和安全性，尤其是全身治疗转移和腹腔扩散肿瘤，为恶性肿瘤治疗提供一种新的方法。

5型腺病毒H101作为世界上首个治疗头颈肿瘤的溶肿瘤腺病毒已在中国上市，B亚群腺病毒Ad11与Ad5相比，具有肿瘤细胞内受体表达高，体内中和抗体低和肝毒性低等明显的优势。本研究首先构建了具有肿瘤靶向性和免疫治疗基因结合的新型溶肿瘤病毒，利用细胞模型和转基因动物模型进行体内外抗肿瘤效果和安全性实验，以及其抗肿瘤作用机制。主要发现：成功构建了多个肿瘤靶向性溶肿瘤腺病毒，包括（1）Ad11-Ad5-EP-TD，Ad11-Tel-GFP、Ad11-Tel-mIL21、 Ad11-Tel-hIL21和Ad11-Tel-mIL15R/IL15；（2）Ad11-Tel-mIL15R/IL15能选择性地杀伤肺癌肿瘤细胞，在肿瘤细胞内高表达IL15，在肺癌皮下和转移瘤模型中有很好的抗肿瘤效果；（3）安全性实验表明，Ad11-Tel-mIL15R/IL15不仅可以用于局部肿瘤内注射，还可用于静脉注射，抗肿瘤机制研究发现CD4+和CD8+T细胞起主要的抗肿瘤作用。这种新型的复制选择性腺病毒为人类肿瘤治疗提供了新的方法。