Dombrowsky Carolin Sophie, Happel Dominic, Habermann Jan, Hofmann Sarah, Otmi Sasi, Cohen Benny, Kolmar Harald
Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Peter-Grünberg-Strasse 4, D-64287 Darmstadt, Germany.
Inter-Lab, a Subsidiary of Merck KGaA, South Industrial Area, Yavne 8122004, Israel.
Antibodies (Basel). 2024 May 2;13(2):37. doi: 10.3390/antib13020037.
Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells. Functional characterization revealed that the tested antibody has beneficial cytosol-penetrating capabilities and can deliver cargo proteins (up to 70 kDa) to the cytosol. To achieve tumor-specific cell targeting and cargo delivery through conditional activation of the cell-penetrating antibody in the tumor microenvironment, a single-chain Fc fragment (scFv) and a V domain were isolated as masking units. Several in vitro assays demonstrated that fusing the masking protein with a cleavable linker to the cell penetration antibody results in the inactivation of antibody cell binding and internalization. Removal of the mask via MMP-9 protease cleavage, a protease that is frequently overexpressed in the tumor microenvironment (TME), led to complete regeneration of binding and cytosol-penetrating capabilities. Masked and conditionally activated cytosol-penetrating antibodies have the potential to serve as a modular platform for delivering protein cargoes addressing intracellular targets in tumor cells.
目前，抗体的治疗和诊断应用主要限于细胞表面暴露的和细胞外的蛋白质。然而，为克服这些限制，人们已对细胞穿透肽（CPP）以及可穿透胞质溶胶的抗体展开了研究。在此背景下，偶然发现了一种硫酸乙酰肝素蛋白聚糖（HSPG）结合抗体，它最终定位于靶细胞的胞质溶胶中。功能特性分析表明，所测试的抗体具有有益的胞质溶胶穿透能力，并且能够将负载蛋白（高达70 kDa）递送至胞质溶胶。为了通过在肿瘤微环境中对可穿透细胞的抗体进行条件激活来实现肿瘤特异性细胞靶向和负载递送，分离出单链Fc片段（scFv）和V结构域作为掩蔽单元。多项体外试验表明，将掩蔽蛋白与可裂解连接子融合至细胞穿透抗体可导致抗体细胞结合和内化失活。通过MMP-9蛋白酶切割去除掩蔽物（MMP-9蛋白酶在肿瘤微环境（TME）中经常过表达）可使结合和胞质溶胶穿透能力完全恢复。掩蔽且经条件激活的可穿透胞质溶胶的抗体有潜力作为一个模块化平台，用于递送针对肿瘤细胞内靶点的蛋白负载。
