Dombrowsky Carolin Sophie, Geyer Felix Klaus, Zakharchuk Diana, Kolmar Harald
Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Peter-Grünberg-Strasse 4, 64287 Darmstadt, Germany.
Centre for Synthetic Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany.
Mol Ther Oncol. 2025 Jan 2;33(1):200931. doi: 10.1016/j.omton.2024.200931. eCollection 2025 Mar 20.
Although a considerable number of disease-related biomolecular interactions occur in the cytosol, therapeutic and diagnostic application of target-specific binding proteins is largely confined to surface-exposed or extracellular targets. Therefore, protein-cargo delivery approaches, including cell-penetrating peptides and cytosol-penetrating antibodies, are being explored to overcome this limitation. In this context, we have developed a modular approach for cytosolic penetration of tumor cells based on bispecific antibodies containing a masked cytosol-penetrating Fab on one arm and a tumor-targeting scFv linked via an endosomal cleavable linker on the other arm. The relevance of the antigen-specific binding, internalization, and cytosolic cargo delivery was demonstrated in several assays using different cell lines with anti-B7-H3 scFv, the well-characterized trastuzumab (HER2), and inotuzumab (CD22) as examples. In addition, presence of the masking moiety to prevent non-specific surface binding, as well as the activation of cytosol-penetrating capabilities in the tumor microenvironment upon release by tumor-specific proteases was confirmed using the catalytic domain of exotoxin as model cargo for cytosol delivery. Tumor microenvironment-dependent as well as tumor-associated antigen-specific cytosol-penetrating antibodies of the type developed here have the potential to serve as a modular platform to deliver macromolecular cargoes for addressing intracellular targets in tumor cells.
尽管相当数量的疾病相关生物分子相互作用发生在细胞质中，但靶向特异性结合蛋白的治疗和诊断应用主要局限于表面暴露或细胞外靶点。因此，正在探索包括细胞穿透肽和细胞质穿透抗体在内的蛋白质-货物递送方法来克服这一限制。在此背景下，我们基于双特异性抗体开发了一种肿瘤细胞细胞质穿透的模块化方法，该双特异性抗体的一条臂含有被屏蔽的细胞质穿透Fab片段，另一条臂通过内体可裂解连接子连接肿瘤靶向单链抗体片段（scFv）。使用不同细胞系，以抗B7-H3 scFv、特征明确的曲妥珠单抗（HER2）和伊奈妥单抗（CD22）为例，在多个试验中证明了抗原特异性结合、内化和细胞质货物递送的相关性。此外，使用外毒素催化结构域作为细胞质递送的模型货物，证实了存在屏蔽部分以防止非特异性表面结合，以及肿瘤特异性蛋白酶释放后在肿瘤微环境中细胞质穿透能力的激活。这里开发的这种类型的肿瘤微环境依赖性以及肿瘤相关抗原特异性细胞质穿透抗体有潜力作为一个模块化平台，用于递送大分子货物以靶向肿瘤细胞内的靶点。
