Uusi-Kerttula Hanni, Legut Mateusz, Davies James, Jones Rachel, Hudson Emma, Hanna Louise, Stanton Richard J, Chester John D, Parker Alan L
1Institutes of Cancer and Genetics, Cardiff University, Cardiff CF14 4XN, United Kingdom.
2Institutes of Infection and Immunity, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom.
Hum Gene Ther. 2015 May;26(5):320-9. doi: 10.1089/hum.2015.015.
Oncolytic virotherapies based on adenovirus 5 (Ad5) hold promise as adjunctive cancer therapies; however, their efficacy when delivered systemically is hampered by poor target cell specificity and preexisting anti-Ad5 immunity. Ovarian cancer represents a promising target for virotherapy, since the virus can be delivered locally into the peritoneal cavity. Both epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) are overexpressed in the majority of human tumors, including ovarian cancer. To generate adenoviral vectors with improved tumor specificity, we generated a panel of Ad5 vectors with altered tropism for EGFR and FGFR, rather than the natural Ad5 receptor, hCAR. We have included mutations within AB loop of the viral fiber knob (KO1 mutation) to preclude interaction with hCAR, combined with insertions in the HI loop to incorporate peptides that bind either EGFR (peptide YHWYGYTPQNVI, GE11) or FGFR1 (peptides MQLPLAT, M*, and LSPPRYP, LS). Viruses were produced to high titers, and the integrity of the fiber protein was validated by Western blotting. The KO1 mutation efficiently ablated hCAR interactions, and significantly increased transduction was observed in hCAR(low)/EGFR(high) cell lines using Ad5.GE11, while transduction levels using Ad5.M* or Ad5.LS were not increased. In the presence of physiological concentrations of human blood clotting factor X (hFX), significantly increased levels of transduction via the hFX-mediated pathway were observed in cell lines, but not in primary tumor cells derived from epithelial ovarian cancer (EOC) ascites samples. Ad5-mediated transduction of EOC cells was completely abolished by the presence of 2.5% serum from patients, while, surprisingly, incorporation of the GE11 peptide resulted in significant evasion of neutralization in the same samples. We thus speculate that incorporation of the YHWYGYTPQNVI dodecapeptide within the fiber knob domain may provide a novel means of circumventing preexisting Ad5 immunity that warrants further investigation.
基于腺病毒5（Ad5）的溶瘤病毒疗法有望成为辅助癌症治疗手段；然而，全身给药时其疗效受到靶细胞特异性差和预先存在的抗Ad5免疫的阻碍。卵巢癌是病毒疗法的一个有前景的靶点，因为病毒可以局部递送至腹腔。表皮生长因子受体（EGFR）和成纤维细胞生长因子受体1（FGFR1）在包括卵巢癌在内的大多数人类肿瘤中均过度表达。为了生成具有更高肿瘤特异性的腺病毒载体，我们构建了一组对EGFR和FGFR具有改变嗜性的Ad5载体，而非天然的Ad5受体hCAR。我们在病毒纤维钮的AB环内引入了突变（KO1突变）以阻止与hCAR相互作用，并在HI环中进行插入以掺入与EGFR（肽YHWYGYTPQNVI，GE11）或FGFR1（肽MQLPLAT，M*，以及LSPPRYP，LS）结合的肽。病毒产生了高滴度，并且通过蛋白质印迹法验证了纤维蛋白的完整性。KO1突变有效地消除了hCAR相互作用，并且使用Ad5.GE11在hCAR（低）/EGFR（高）细胞系中观察到转导显著增加，而使用Ad5.M*或Ad.LS时转导水平并未增加。在存在生理浓度的人凝血因子X（hFX）的情况下，在细胞系中观察到通过hFX介导的途径转导水平显著增加，但在源自上皮性卵巢癌（EOC）腹水样本的原代肿瘤细胞中未观察到。患者2.5%的血清会完全消除Ad5介导的EOC细胞转导，而令人惊讶的是，掺入GE11肽导致在相同样本中显著逃避中和作用。因此，我们推测在纤维钮结构域内掺入YHWYGYTPQNVI十二肽可能提供一种规避预先存在的Ad5免疫的新方法，值得进一步研究。
