溶瘤病毒是一种有前途的技术，可以攻击癌细胞并招募免疫细胞到肿瘤部位。由于Lipocalin-2受体（LCN2R）存在于大多数癌细胞上，我们使用它的配体LCN2来针对溶瘤腺病毒（Ads）靶向癌细胞。因此，我们将一个DARPin适配器融合到Ad5 (knob5) 的顶部来与LCN2结合，以重新定向病毒，以分析这种新的靶向方法的基本特性。该适配器在稳定表达LCN2R的中国仓鼠卵巢（CHO）细胞和20种癌细胞系（CCLs）中使用Ad5病毒载体编码荧光素酶和绿色荧光蛋白进行了体外测试。LCN2适配器（LA）的荧光素酶测定显示与阻塞适配器（BA）相比，在表达LCN2R和不表达LCN2R的CHO细胞中，感染率提高了10倍。大多数CCLs显示出与BA绑定的病毒相比，LA-bound病毒的病毒摄取量增加了，并且对于五种CCLs，病毒摄取量与未改修的Ad5相当。流式细胞术和六方体免疫染色也显示出，在大多数测试的CCLs中，LA-bound病毒的摄取量增加，而BA-bound病毒的摄取量减少。在3D细胞培养模型中研究了病毒扩散，结果九种CCLs显示出LA-bound病毒的荧光信号比BA-bound病毒更早地增加。从机理上讲，我们发现，LCN2适配器仅在配体Enterobactin (Ent)缺失且独立于铁的情况下增加病毒的摄取量。综上，我们表征了一种新的基于DARPin的系统，能增强病毒的摄取，展示了未来溶瘤病毒疗法的潜力。

Oncolytic viruses are a promising technology to attack cancer cells and to recruit immune cells to the tumor site. Since the Lipocalin-2 receptor (LCN2R) is expressed on most cancer cells, we used its ligand LCN2 to target oncolytic adenoviruses (Ads) to cancer cells. Therefore, we fused a DARPin adapter binding the knob of Ad type 5 (knob5) to LCN2 to retarget the virus towards LCN2R with the aim of analyzing the basic characteristics of this novel targeting approach. The adapter was tested in vitro with Chinese Hamster Ovary (CHO) cells stably expressing the LCN2R and on 20 cancer cell lines (CCLs) using an Ad5 vector encoding luciferase and green fluorescent protein (GFP). Luciferase assays with the LCN2 adapter (LA) showed 10-fold higher infection compared to blocking adapter (BA) in CHO cells expressing LCN2R and in cells not expressing the LCN2R. Most CCLs showed an increased viral uptake of LA-bound virus compared to BA-bound virus and for five CCLs viral uptake was comparable to unmodified Ad5. Flow cytometry and hexon immunostainings also revealed increased uptake of LA-bound Ads compared with BA-bound Ads in most tested CCLs. Virus spread was studied in 3D cell culture models and nine CCLs showed increased and earlier fluorescence signals for LA-bound virus compared to BA-bound virus. Mechanistically we show that the LCN2 adapter increases viral uptake only in the absence of its ligand Enterobactin (Ent) and independently of iron. Together, we characterized a novel DARPin-based system resulting in enhanced uptake demonstrating potential for future oncolytic virotherapy.