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Currently, 7 of the top 10 selling drugs are biologics, and all of them are proteins. Their large size, structural complexity, and molecular diversity often results in surfaces capable of potent and selective recognition of receptors that challenge, or evade, traditional small molecules. However, most proteins do not penetrate the lipid bilayer exterior of mammalian cells. This severe limitation dramatically limits the number of disease-relevant receptors that proteins can target and modulate. Given the major role proteins play in modern medicine, and the magnitude of this limitation, it is unsurprising that an enormous amount of effort has been dedicated to overcoming this pesky impediment. In this article, we summarize and evaluate current approaches for intracellular delivery of exogenous proteins to mammalian cells and, in doing so, aim to illuminate fertile ground for future discovery in this critical area of research.

By virtue of their size, functional group diversity, and complex structure, proteins can often recognize and modulate disease-relevant macromolecules that present a challenge to small-molecule reagents. Additionally, high-throughput screening and evolution-based methods often make the discovery of new protein binders simpler than the analogous small-molecule discovery process. However, most proteins do not cross the lipid bilayer membrane of mammalian cells. This largely limits the scope of protein therapeutics and basic research tools to those targeting disease-relevant receptors on the cell surface or extracellular matrix. Previously, researchers have shown that cationic resurfacing of proteins can endow cell penetration. However, in our experience, many proteins are not amenable to such extensive mutagenesis. Here, we report that nanobodies-a small and stable protein that can be evolved to recognize virtually any disease-relevant receptor-are amenable to cationic resurfacing, which results in cell internalization. Once internalized, these nanobodies access the cytosol. Polycationic resurfacing does not appreciably alter the structure, expression, and function (target recognition) of a previously reported GFP-binding nanobody, and multiple nanobody scaffolds are amenable to polycationic resurfacing. Given this, we propose that polycationic resurfaced cell-penetrating nanobodies might represent a general scaffold for intracellularly targeted protein drug discovery.

Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blot are common bioanalytical techniques. Successful execution traditionally requires the use of one or more commercially available antibody-small-molecule dye, or antibody-reporter protein conjugates that recognize relatively short peptide tags (