A Conditionally Activated Cytosol-Penetrating Antibody for TME-Dependent Intracellular Cargo Delivery

• Published in: Antibodies
• Main Authors: Carolin Sophie Dombrowsky, Dominic Happel, Jan Habermann, Sarah Hofmann, Sasi Otmi, Benny Cohen, Harald Kolmar
• Format: Article
• Language: English
• Published: MDPI AG 2024-05-01
• Subjects: cytosol-penetrating antibody; cytosolic delivery; endosomal escape; masked cytosol-penetrating antibody; MMP-9; TME
• Online Access: https://www.mdpi.com/2073-4468/13/2/37

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_L 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.