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A strategy for the bioorthogonal immobilization of proteins onto commercially available filter paper is presented. Recently, a two-step approach has been described that relies on covalent immobilization of a linker molecule to paper, followed by enzyme-mediated conjugation of a protein of interest containing an enzyme-recognition tag. Here, this strategy was expanded by evaluating four different chemical and chemoenzymatic reactions and investigating paper loading efficiency and orthogonality. Enhanced green fluorescent protein (EGFP) was used as a model protein to allow quantification of protein loading via fluorescence imaging. Two approaches were identified that showed significantly increased loading efficiencies compared with the previously applied conjugation strategy. Additionally, all four methods were proven orthogonal to each other, allowing simultaneous immobilization of a mixture of proteins to a premodified assembly of two paper sheets.

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.