Proteins have the capacity to treat a multitude of diseases both as therapeutics and as drug carriers due to their complex functional properties, specificity toward binding partners, biocompatibility, and programmability. Despite this, native proteins often require assistance to target diseased tissue due to poor pharmacokinetic properties and membrane impermeability. Functionalizing therapeutic proteins and drug carriers through direct conjugation of delivery moieties can enhance delivery capabilities. Traditionally, this has been accomplished through bioconjugation methods that have little control over the location or orientation of the modification, leading to highly heterogeneous products with varying activity. A multitude of promising site-specific protein conjugation methods have been developed to allow more tailorable display of delivery moieties and thereby enhance protein activity, circulation properties, and targeting specificity. Here, we focus on three particularly promising site-specific bioconjugation techniques for protein delivery: unnatural amino acid incorporation, Sortase-mediated ligation, and SpyCatcher/SpyTag chemistry. In this review, we highlight the promise of site-specific bioconjugation for targeted drug delivery by summarizing impactful examples in literature, considering important design principles when constructing bioconjugates, and discussing our perspectives on future directions.

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Naturally occurring protein nanocages are promising drug carriers because of their uniform size and biocompatibility. Engineering efforts have enhanced the delivery properties of nanocages, but cell specificity and high drug loading remain major challenges. Herein, we fused the SpyTag peptide to the surface of engineered E2 nanocages to enable tunable nanocage decoration and effective E2 cell targeting using a variety of SpyCatcher (SC) fusion proteins. Additionally, the core of the E2 nanocage incorporated four phenylalanine mutations previously shown to allow hydrophobic loading of doxorubicin and pH-responsive release in acidic environments. We functionalized the surface of the nanocage with a highly cell-specific epidermal growth factor receptor (EGFR)-targeting protein conjugate, 4GE11-mCherry-SC, developed previously in our laboratories by employing unnatural amino acid (UAA) protein engineering chemistries. Herein, we demonstrated the benefits of this engineered protein nanocage construct for efficient drug loading, with a straightforward method for removal of the unloaded drug through elastin-like polypeptide-mediated inverse transition cycling. Additionally, we demonstrated approximately 3-fold higher doxorubicin internalization in inflammatory breast cancer cells compared to healthy breast epithelial cells, leading to targeted cell death at concentrations below the IC50 of free doxorubicin. Collectively, these results demonstrated the versatility of our UAA-based EGFR-targeting protein construct to deliver a variety of cargoes efficiently, including engineered E2 nanocages capable of site-specific functionalization and doxorubicin loading.

Protein therapeutics offer enormous clinical impact in treating a variety of diseases by offering high selectivity with limited off-target effects. However, delivery challenges severely reduce functional proteins from reaching their...