Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, 52074 Aachen, Germany.
Curr Pharm Des. 2013;19(30):5429-36. doi: 10.2174/13816128113199990405.
Although current cancer treatment strategies are highly aggressive, they are often not effective enough to destroy the collectivity of malignant cells. The residual tumor cells that survived the first-line treatment may continue to proliferate or even metastasize. Therefore, the development of novel more effective strategies to specifically eliminate also single cancer cells is urgently needed. In this respect, the development of antibody-based therapeutics, in particular example immunotoxins, has attracted broad interest. Since the internalization of immunotoxins is essential for their cytotoxic effectivity, it is of crucial importance to study their internalization behavior to assess the potential for their therapeutic use. In this study, we determined the internalization behavior of four different single-chain fragments variable (scFv) when binding to the corresponding target antigen as expressed on solid or non-solid tumor cell lines. The scFvs were recombinantly fused to the SNAP-tag, an engineered variant of the human repair enzyme O(6)-alkylguanine-DNA alkyltransferase that covalently reacts with benzylguanine derivatives. Since a large number of highly sensitive organic fluorescent dyes are already available or can easily be derivatized to react with the self-labeling SNAP-tag, this system provides versatile applications for imaging of intraand extracellular compartments of living cells. The fusion proteins were coupled to SNAP-surface(®) Alexa Fluor(®) 488 or SNAP-surface(®) Alexa Fluor(®) 647 and binding as well as internalization was monitored by flow cytometry and confocal microscopy, respectively. Depending on the respective target antigen, we could distinguish between slow and rapid internalization behavior. Moreover, we detected increased internalization rate for bivalent scFv constructs. Our approach allows for rapid and early stage evaluation of the internalization characteristics of new antibodies designated for further therapeutic development.
虽然目前的癌症治疗策略非常激进，但往往不足以彻底消灭恶性细胞群体。一线治疗中幸存的肿瘤细胞可能会继续增殖，甚至转移。因此，迫切需要开发新型更有效的策略来特异性地消除单个癌细胞。在这方面，基于抗体的治疗方法的发展，特别是免疫毒素，引起了广泛的关注。由于免疫毒素的内化对于其细胞毒性效力至关重要，因此研究其内化行为对于评估其治疗用途至关重要。在这项研究中，我们确定了四个不同的单链片段可变区 (scFv) 与相应的靶抗原结合时的内化行为，这些 scFv 表达在固体或非固体肿瘤细胞系上。scFv 被重组融合到 SNAP 标签上，SNAP 标签是人类修复酶 O(6)-烷基鸟嘌呤-DNA 烷基转移酶的工程变体，可与苯甲基鸟嘌呤衍生物共价反应。由于已经有大量高度敏感的有机荧光染料可用，或者可以很容易地衍生为与自标记 SNAP 标签反应，因此该系统为活细胞内外区室的成像提供了多种应用。融合蛋白与 SNAP-surface(®) Alexa Fluor(®) 488 或 SNAP-surface(®) Alexa Fluor(®) 647 偶联，通过流式细胞术和共聚焦显微镜分别监测结合和内化。根据各自的靶抗原，我们可以区分缓慢和快速的内化行为。此外，我们检测到二价 scFv 构建体的内化率增加。我们的方法允许快速和早期评估指定用于进一步治疗开发的新抗体的内化特性。
