Ding Haozhong, Altai Mohamed, Yin Wen, Lindbo Sarah, Liu Hao, Garousi Javad, Xu Tianqi, Orlova Anna, Tolmachev Vladimir, Hober Sophia, Gräslund Torbjörn
Department of Protein Science, KTH Royal Institute of Technology, Roslagstullsbacken 21, 114 17 Stockholm, Sweden.
Department of Oncology and Pathology, Barngatan 4, Lund University, 222 42 Lund, Sweden.
Pharmaceutics. 2020 Apr 24;12(4):391. doi: 10.3390/pharmaceutics12040391.
The human epidermal growth factor receptor 2 (HER2) is a clinically validated target for cancer therapy, and targeted therapies are often used in regimens for patients with a high HER2 expression level. Despite the success of current drugs, a number of patients succumb to their disease, which motivates development of novel drugs with other modes of action. We have previously shown that an albumin binding domain-derived affinity protein with specific affinity for HER2, ADAPT, can be used to deliver the highly cytotoxic protein domain PE25, a derivative of Pseudomonas exotoxin A, to HER2 overexpressing malignant cells, leading to potent and specific cell killing. In this study we expanded the investigation for an optimal targeting domain and constructed two fusion toxins where a HER2-binding affibody molecule, Z, or the dual-HER2-binding hybrid Z-ADAPT were used for cancer cell targeting. We found that both targeting domains conferred strong binding to HER2; both to the purified extracellular domain and to the HER2 overexpressing cell line SKOV3. This resulted in fusion toxins with high cytotoxic potency toward cell lines with high expression levels of HER2, with EC values between 10 and 100 pM. For extension of the plasma half-life, an albumin binding domain was also included. Intravenous injection of the fusion toxins into mice showed a profound influence of the targeting domain on biodistribution. Compared to previous results, with ADAPT as targeting domain, Z gave rise to further extension of the plasma half-life and also shifted the clearance route of the fusion toxin from the liver to the kidneys. Collectively, the results show that the targeting domain has a major impact on uptake of PE25-based fusion toxins in different organs. The results also show that PE25-based fusion toxins with high affinity to HER2 do not necessarily increase the cytotoxicity beyond a certain point in affinity. In conclusion, Z has the most favorable characteristics as targeting domain for PE25.
人表皮生长因子受体2（HER2）是癌症治疗中经过临床验证的靶点，靶向治疗常用于HER2表达水平高的患者治疗方案中。尽管现有药物取得了成功，但仍有许多患者死于疾病，这推动了具有其他作用方式的新型药物的研发。我们之前已经表明，一种对HER2具有特异性亲和力的白蛋白结合域衍生亲和蛋白ADAPT，可用于将高细胞毒性蛋白结构域PE25（一种铜绿假单胞菌外毒素A的衍生物）递送至HER2过表达的恶性细胞，从而实现强效且特异性的细胞杀伤。在本研究中，我们扩展了对最佳靶向结构域的研究，并构建了两种融合毒素，其中使用HER2结合亲和体分子Z或双HER2结合杂合体Z - ADAPT进行癌细胞靶向。我们发现这两个靶向结构域都能与HER2产生强结合，无论是与纯化的细胞外结构域还是与HER2过表达细胞系SKOV3。这导致融合毒素对HER2高表达细胞系具有高细胞毒性，其半数有效浓度（EC）值在10至100皮摩尔之间。为了延长血浆半衰期，还加入了白蛋白结合域。将融合毒素静脉注射到小鼠体内显示，靶向结构域对生物分布有深远影响。与之前以ADAPT作为靶向结构域的结果相比，Z使血浆半衰期进一步延长，并且还将融合毒素的清除途径从肝脏转移至肾脏。总体而言，结果表明靶向结构域对基于PE25的融合毒素在不同器官中的摄取有重大影响。结果还表明，对HER2具有高亲和力的基于PE25的融合毒素不一定会在亲和力超过某一点后增加细胞毒性。总之,Z作为PE25的靶向结构域具有最有利的特性。
