Controlled EGFR ligand display for tunable targeted intracellular delivery of cancer suicide enzymes
June 5-9, 2018
Advances in molecular engineering have led to customizable proteins for a multitude of clinical applications, leading to the rapid growth of the protein pharmaceutical market. Proteins can be advantageous compared to other treatment methods because of their functional complexities and high specificity that cannot be mimicked by small molecule drugs. Although there has been great interest and investment in advanced protein therapeutics, a majority of marketed proteins continue to have extracellular targets, despite the therapeutic relevance for intracellular protein therapies. Engineering efforts to improve cytosolic protein delivery often rely on modifying proteins through direct conjugation of polymers and peptides using reactive residues on amino acids. While this method has shown some success, the inability to modify a specific site within a protein can significantly hinder pharmacological action.Additionally, such approaches do not offer control over design variables that can be important determinants of targeting efficacy, such as ligand clustering.
Previous work has demonstrated the ability to site-specifically insert biorthogonal reactive residues into proteins through unnatural amino acid incorporation, enabling direct protein conjugation with simple ‘click’ chemistries. Such an approach could be applied to protein therapeutics to explore the effect arrangement of delivery molecules has on protein bioactivity and intracellular delivery capabilities. In our work, we have demonstrated application of this approach for conjugation of epidermal growth factor receptor (EGFR) targeting peptides in fluorescent proteins. By varying EGFR peptide arrangements we have demonstrated the ability to tune cellular internalization in inflammatory breast cancer (IBC) cells. Furthermore, this system has been adapted for delivery of a cancer suicide enzyme to enable IBC-targeted cell death through prodrug activation. Through this approach, we have identified the importance of ligand display for targeted protein delivery and applied these finding to enhance enzyme delivery to IBC cells. Future efforts will refine the efficacy of this method though incorporation of endosomal escaping peptides and hydrophilic polymers to address additional challenges associated with in vivo intracellular protein delivery.
Rachel Lieser, Wilfred Chen, and Millicent Sullivan, "Controlled EGFR ligand display for tunable targeted intracellular delivery of cancer suicide enzymes" in "Nanotechnology in Medicine II: Bridging Translational in vitro and in vivo Interfaces", Millicent Sullivan, PhD, University of Delaware, USA Josué Sznitman, Dr. Sc., Technion-Israel Institute of Technology, Israel Lola Eniola-Adefeso, PhD, University of Michigan, USA Srivatsan Kidambi, PhD, University of Nebraska - Lincoln, USA Eds, ECI Symposium Series, (2018). http://dc.engconfintl.org/nanotech_med_ii/8