Proteins have emerged as a new class of therapeutics in the past several decades due to breakthroughs in molecular engineering that allow researchers to customize proteins for a multitude of clinical applications. However, despite great interest and investment in advanced protein therapeutics, most FDA-approved proteins have extracellular targets, despite the existence of multiple diseases that could be treated through intracellular protein therapies. Engineering efforts to address delivery limitations often rely on modifying proteins through direct conjugation of polymers and peptides using reactive residues on naturally occurring amino acids. The key shortcoming of this method is the inability to modify a specific site within a protein, which can significantly reduce pharmacological action. Additionally, such approaches do not offer control over variables such as ligand clustering, which is an important determinant of targeting efficacy.<br/><br/>Unnatural amino acid (UAA) incorporation provides a method to modify proteins with site-specific, biorthogonal reactive moieties through nonsense codon replacement. This method of modification not only allows site-specific functionalization of therapeutics for improved bioactivity, but also allows exploration of the arrangement of delivery molecules on proteins for optimal intracellular delivery. Previous work has demonstrated the ability to incorporate biorthogonal chemistries into model proteins through UAA incorporation, enabling modification with simple ‘click’ chemistry techniques. In our work, we demonstrate application of this approach for conjugation of epidermal growth factor receptor (EGFR) targeting peptides in fluorescent proteins, using varying EGFR peptide arrangements to control cellular internalization in inflammatory breast cancer (IBC) cells. Furthermore, we demonstrate the ability to adapt this system for delivery of a suicide enzyme to enable IBC-targeted cell death through prodrug application and conversion. Through this approach, we have identified an ideal ligand display for enzyme delivery to IBC cells. Future work will refine the efficacy of the approach via incorporation of endosomal escaping peptides and hydrophilic polymers, enabling tailorable intracellular protein delivery in vivo.

Controlled EGFR ligand display on cancer suicide enzymes for targeted intracellular delivery

255th National Meeting (2018)

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