Development of an αᵥβ₆-Binding Peptide for In Vivo Applications: Modulation of Serum Stability and Biodistribution
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Targeted delivery of imaging and therapeutic agents to tumors improves detection, characterization, and treatment of many types of cancers. Peptides are capable of efficient and selective delivery of a variety of cargoes to their target cells and have been in development as targeting ligands for a variety of pathologies for several years. The H2009.1 peptide binds to αᵥβ₆, an integrin expressed in 54% of non-small cell lung cancers (NSCLC), 80-100% of head and neck carcinomas, and 37% of colorectal cancers. This peptide binds and is internalized into αᵥβ₆ positive cell lines, and successfully delivers cargo to xenograft tumors. This dissertation presents the optimization of this peptide for in vivo applications (i.e. improvement of serum stability and reduction of renal accumulation). The N-termini of tetrameric H2009.1 peptide were capped by acetylation or by unnatural amino acids. This capping significantly increased the half-life of H2009.1 tetramer in human serum. In vivo near-infrared fluorescence (NIRF) imaging shows that N-terminal capping also decreases kidney accumulation of this peptide at short time periods while maintaining targeting to the tumor for up to 72 hrs. To further decrease off-target accumulation, the valency of the targeting ligands was reduced from a tetramer to a dimer. The dimeric H2009.1 peptide produces similar targeting capabilities compared to the tetrameric peptide, with decreased accumulation in the liver, lung, and kidney. The optimal peptide for long-term targeting of therapeutics to αᵥβ₆-positive NSCLCs is the AcH2009.1 dimer as it gives the highest accumulation and retention in target tumors while avoiding kidney and lung tissue. The best peptide for short-term applications is the AcD-Leu H2009.1 dimer, which differentiates well between αᵥβ₆-positive and negative tumors and clears rapidly from the animal. While further work is necessary to bring the H2009.1 peptide into clinical use, the results and experiences of this thesis project have provided critical insights into the in vivo optimization of this and many other peptide targeting ligands.