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PROGRAM | Chemical Engineering

Addressing Intracellular Protein Delivery Challenges with Site-Specific Protein Conjugation Strategies for Breast Cancer Treatment

By: Rachel Lieser Chair: Wilfred Chen Co-Chair: Millicent Sullivan


Proteins have the capacity to treat a multitude of diseases both as therapeutics and drug carriers due to their complex functional properties, specificity toward binding partners, biocompatibility, and programmability. Despite this, native proteins often require active strategies to target diseased tissue due to membrane impermeability. Additionally, proteins are often uptaken through endocytosis, so accessing the cytosol or other subcellular compartments requires internalized protein to escape the endosome. As a result, intracellular drug candidates make up less than 5% of protein therapeutics entering clinical development despite having immense therapeutic potential to treat a multitude of diseases including cancer. Functionalizing therapeutic proteins and drug carriers through direct conjugation of delivery moieties can enhance delivery capabilities. Traditionally, this has been accomplished using reactive residues (e.g. lysines) within the protein sequence, or genetic fusion to the protein termini. While both methods have the capacity to enhance various aspects of delivery, the inability to chemically modify proteins with site-specificity often leads to highly heterogeneous products with varying levels of activity. Additionally, such approaches do not offer control over variables such as ligand clustering, which can be an important determinant of targeting efficacy.

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. In this thesis we focus on two particularly promising site-specific bioconjugation techniques to enhance intracellular protein delivery: unnatural amino acid (UAA) incorporation and SpyCatcher/SpyTag chemistry. To this end, we have developed a versatile protein-peptide conjugate capable of targeting cancer cells that overexpress epidermal growth factor receptor (EGFR) for delivery of a multitude of cargos.

Previous work has demonstrated the ability to insert biorthogonal reactive residues into proteins through unnatural amino acid (UAA) incorporation, enabling protein conjugation with simple ‘click’ chemistries. Here, we describe a strategy to site-specifically conjugate delivery moieties to therapeutic proteins through UAA incorporation, to explore the effect of EGFR-targeted ligand valency and spacing on internalization of proteins in EGFR-overexpressing inflammatory breast cancer (IBC) cells. Results demonstrate the importance of controlling ligand display on proteins for robust active targeting. In particular, high EGFR ligand valency and clustering was associated with enhanced IBC internalization compared to healthy breast epithelial cells. Furthermore, this EGFR-targeted conjugate was adapted for plug-and-play cargo incorporation with SpyCatcher/SpyTag chemistry. This approach was used to deliver a cancer prodrug converting enzyme and engineered protein nanocages loaded with small molecule chemotherapeutics for IBC-targeted cell death.

Finally, we developed a simple fusion modification strategy to incorporate four endosomolytic peptides, Aurein 1.2, GALA, HA2, and L17E, onto the EGFR-targeted protein conjugate, and evaluated the ability of the peptide modifications to trigger endosomal escape while maintaining EGFR specificity. While all peptides exhibit membrane lytic properties, only Aurein 1.2 and GALA maintained EGFR specificity while also providing moderate endosomal escape capabilities. These results demonstrate the importance of endosomolytic peptide selection when designing targeted therapies and further expands the range of cargos that can be delivered with our EGFR-targeted protein conjugate.

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