To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass.

中文翻译：

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同时使用HER2 / neu或EpCAM作为目标受体，通过双特异性溶瘤性麻疹病毒增强裂解作用。

为了将溶瘤性麻疹病毒（MV）靶向肿瘤，我们利用了设计的锚蛋白重复蛋白（DARPins）的结合特异性。这些DARPin-MV具有高的肿瘤选择性，同时保持了出色的溶瘤效力。DARPins的稳定性，体积小和功效使同时产生针对肿瘤标志物HER2 / neu和癌症干细胞（CSC）标志物EpCAM的MVs成为可能。为了优化，连接两个DARPins的链接器在灵活性和长度上有所不同。柔韧性对融合助手的活动没有影响，而长度却有影响。具有双特异性MV-H的MV具有遗传稳定性，并显示出所需的双靶特异性。在体外，取决于靶细胞，双特异性MV的溶细胞活性优于单靶病毒或具有可比性。体内，在特异性原位卵巢癌模型中验证了双特异性病毒的治疗功效，揭示了肿瘤块的有效减少。最后，在不同肿瘤细胞的共培养物中证明了双特异性靶向的能力，从而在体外模拟了肿瘤异质性，更紧密地反映了真实的肿瘤。在此，双特异性优于单特异性病毒。因此，基于DARPin的靶向域允许产生具有双重特异性的有效溶瘤病毒，具有处理抗原表达的肿瘤内变异并同时靶向CSC和大块肿瘤的潜力。在不同肿瘤细胞的共培养物中证明了双特异性靶向的能力，从而在体外模拟了肿瘤异质性，更紧密地反映了真实的肿瘤。在此，双特异性优于单特异性病毒。因此，基于DARPin的靶向域允许产生具有双重特异性的有效溶瘤病毒，具有处理抗原表达的肿瘤内变异并同时靶向CSC和大块肿瘤的潜力。在不同肿瘤细胞的共培养物中证明了双特异性靶向的能力，从而在体外模拟了肿瘤异质性，更紧密地反映了真实的肿瘤。在此，双特异性优于单特异性病毒。因此，基于DARPin的靶向域允许产生具有双重特异性的有效溶瘤病毒，具有处理抗原表达的肿瘤内变异并同时靶向CSC和大块肿瘤的潜力。