靶向癌症治疗是目前已建立的癌症治疗的一种有前景的替代方案，旨在选择性地杀死癌细胞，同时保护健康组织。因此，分子靶向剂，例如单克隆抗体，被用来特异性结合癌细胞表面标志物。尽管这些药物在临床上取得了巨大的成功，但仍然存在局限性，例如肿瘤渗透性低和脱靶效应。为了克服这一限制，我们设计了由 ADAPT6 和辣根过氧化物酶 (HRP) 两种蛋白组成的新型融合蛋白。癌细胞靶向是通过小支架蛋白 ADAPT6 实现的，该蛋白被设计为特异性结合人表皮生长因子受体 2 (HER2)，这是一种在各种癌症类型中过度表达的细胞表面标记物，而 HRP 酶则氧化无毒的前药 indole-3-乙酸（IAA）会导致自由基的形成，从而对癌细胞产生细胞毒性作用。 ADAPT6-HRP 融合蛋白仍然存在对 HER2 的高亲和力以及 HRP 的酶活性。此外，使用 HER2 阳性 SKOV-3 细胞进行的体外细胞毒性测定揭示了融合蛋白相对于游离 HRP 的明显优势，因为融合蛋白直接与癌细胞结合，从而持续杀死细胞。这种结合 ADAPT6 和 HRP 的新策略代表了一种有前景的方法，也是靶向癌症治疗的抗体偶联的可行替代方案。版权所有 © 2024 作者。由 Elsevier B.V. 出版。保留所有权利。

Targeted cancer therapy is a promising alternative to the currently established cancer treatments, aiming to selectively kill cancer cells while sparing healthy tissues. Hereby, molecular targeting agents, such as monoclonal antibodies, are used to bind to cancer cell surface markers specifically. Although these agents have shown great clinical success, limitations still remain such as low tumor penetration and off-target effects. To overcome this limitation, novel fusion proteins comprised of the two proteins ADAPT6 and Horseradish Peroxidase (HRP) were engineered. Cancer cell targeting is hereby enabled by the small scaffold protein ADAPT6, engineered to specifically bind to human epidermal growth factor receptor 2 (HER2), a cell surface marker overexpressed in various cancer types, while the enzyme HRP oxidizes the nontoxic prodrug indole-3-acetic acid (IAA) which leads to the formation of free radicals and thereby to cytotoxic effects on cancer cells. The high affinity to HER2, as well as the enzymatic activity of HRP, were still present for the ADAPT6-HRP fusion proteins. Further, in vitro cytotoxicity assay using HER2-positive SKOV-3 cells revealed a clear advantage of the fusion proteins over free HRP by association of the fusion proteins directly to the cancer cells and therefore sustained cell killing. This novel strategy of combining ADAPT6 and HRP represents a promising approach and a viable alternative to antibody conjugation for targeted cancer therapy.Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.