Measles virus (MeV) is naturally cytolytic by extensive cell-to-cell fusion. Vaccine-derived MeV is toxic for cancer cells and is clinically tested as oncolytic virus. To combine the potential of MeV with enhanced safety, different targeting strategies have been described. We generated a receptor-targeted MeV by using receptor-blind viral attachment protein genetically fused to designed ankyrin repeat protein (DARPin) binding domains specific for the epidermal growth factor receptor (EGFR). To reduce on-target toxicity for EGFR⁺ healthy cells, we used an engineered viral fusion protein activatable by tumor-associated matrix metalloproteases (MMPs) for additional protease targeting. The dual-targeted virus replicated exclusively on EGFR⁺/MMP⁺ tumor cells but was safe on healthy EGFR⁺ target cells, primary human keratinocytes. Nevertheless, glioblastoma and other tumor cells were efficiently killed by all targeted viruses, although replication and oncolysis were slower for protease-targeted MeV. In vivo, efficacy of EGFR-targeted MeV was virtually unimpaired, whereas also dual-targeted MeV showed significant intra-tumoral spread and efficacy and could be armed with a prodrug convertase. The use of DARPin-domains resulted in potent EGFR-targeted MeV and for the first time effective dual retargeting of an oncolytic virus, further enhancing tumor selectivity. Together with powerful cell-toxic genes, the application as highly tumor-specific platform is promising.

麻疹病毒（MeV）通过广泛的细胞间融合自然被细胞溶解。疫苗衍生的MeV对癌细胞有毒性，并已作为溶瘤病毒进行了临床测试。为了将MeV的潜力与增强的安全性结合起来，已经描述了不同的靶向策略。我们通过使用受体盲型病毒附着蛋白遗传融合到表皮生长因子受体（EGFR）的设计锚蛋白重复蛋白（DARPin）结合域，生成了以受体为目标的MeV。为了降低对EGFR ⁺健康细胞的靶点毒性，我们使用了一种可被肿瘤相关基质金属蛋白酶（MMP）激活的工程化病毒融合蛋白，用于其他蛋白酶靶向。双重靶向病毒仅在EGFR ⁺ / MMP ⁺上复制肿瘤细胞，但对健康的EGFR ⁺靶细胞，原代人角质形成细胞安全。尽管如此，胶质母细胞瘤和其他肿瘤细胞已被所有靶向病毒有效杀死，尽管针对蛋白酶靶向的MeV的复制和溶瘤作用较慢。在体内，以EGFR为靶点的MeV的疗效几乎没有受到损害，而以双重靶点的MeV也显示出显着的肿瘤内扩散和功效，并且可以用前药转化酶进行治疗。DARPin结构域的使用可产生强效的EGFR靶向MeV，并首次有效地将溶瘤病毒双重靶向，从而进一步增强了肿瘤的选择性。连同强大的细胞毒性基因，作为高度肿瘤特异性平台的应用前景广阔。