Abstract
Elastin-like polypeptides (ELP) are versatile protein biopolymers used in drug delivery due to their modular nature, allowing fusion of therapeutics and targeting agents. We previously developed an ELP fusion with vascular endothelial growth factor (VEGF) and demonstrated its therapeutic efficacy in translational swine models of renovascular disease and chronic kidney disease. The goal of the current work was to refine renal targeting and reduce off-target tissue deposition of ELP–VEGF. The ELP–VEGF fusion protein was modified by adding a kidney-targeting peptide (KTP) to the N-terminus. All control proteins (ELP, KTP–ELP, ELP–VEGF, and KTP–ELP–VEGF) were also produced to thoroughly assess the effects of each domain on in vitro cell binding and activity and in vivo pharmacokinetics and biodistribution. KTP–ELP–VEGF was equipotent to ELP–VEGF and free VEGF in vitro in the stimulation of primary glomerular microvascular endothelial cell proliferation, tube formation, and extracellular matrix invasion. The contribution of each region of the KTP–ELP–VEGF protein to the cell binding specificity was assayed in primary human renal endothelial cells, tubular epithelial cells, and podocytes, demonstrating that the VEGF domain induced binding to endothelial cells and the KTP domain increased binding to all renal cell types. The pharmacokinetics and biodistribution of KTP–ELP–VEGF and all control proteins were determined in SKH-1 Elite hairless mice. The addition of KTP to ELP slowed its in vivo clearance and increased its renal deposition. Furthermore, addition of KTP redirected ELP–VEGF, which was found at high levels in the liver, to the kidney. Intrarenal histology showed similar distribution of all proteins, with high levels in blood vessels and tubules. The VEGF-containing proteins also accumulated in punctate foci in the glomeruli. These studies provide a thorough characterization of the effects of a kidney-targeting peptide and an active cytokine on the biodistribution of these novel biologics. Furthermore, they demonstrate that renal specificity of a proven therapeutic can be improved using a targeting peptide.
Highlights
Elastin-like polypeptides (ELP) are a class of protein biopolymers composed of repeating five-amino acid units (VPGxG, where x is any amino acid except proline) with unique physical properties [1] and many advantages as drug carriers [2]
kidney-targeting peptide (KTP)–ELP and KTP–ELP–vascular endothelial growth factor (VEGF) were expressed by bacteria at much lower levels, typically less than 1 mg of protein per liter of bacterial culture
A duplicate gel was probed for VEGF, and the ELP–VEGF and KTP–ELP–VEGF lanes produced strongly reactive bands that matched well to the silver-stained bands, with no VEGF reactivity in the ELP or KTP–ELP lanes
Summary
Elastin-like polypeptides (ELP) are a class of protein biopolymers composed of repeating five-amino acid units (VPGxG, where x is any amino acid except proline) with unique physical properties [1] and many advantages as drug carriers [2]. ELPs are highly soluble in aqueous solution below a distinct transition temperature, and they form coacervates and precipitate above the transition temperature This aggregation process is fully reversible, and the transition temperature at which it occurs can be precisely tuned by changing the hydrophobicity of the guest residue in the VPGxG repeat or by changing the number of repeats [9]. This tunable phase transition makes ELPs extremely versatile as drug delivery platforms via three major strategies: ELPs with transition temperatures below body temperature can be used as slow-release drug depots, ELPs with transition temperatures just above body temperature can be used for thermally targeted drug delivery, and ELPs with high transition temperatures (above body temperature) can be used as soluble protein carriers for therapeutics
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