Michaela received her B.S. in Biochemical Engineering in 2013 from Colorado School of Mines. As a KU graduate student in the pharmaceutical chemistry department, she studies a metal binding tripeptide known as the claMP Tag, which can be incorporated into a targeting protein to create a bioconjugate for the targeted delivery of transition metals. Effective delivery requires stability in the circulating environment, yet degradation inside of diseased cells for metal release. Proteases are a main concern, causing unwanted degradation in the circulating plasma, and characterization of the proteolytic susceptibility of the claMP Tag will determine the use of the claMP Tag as a targeted delivery system.
Design and Characterization of a claMP-Tagged
EGFR Targeting Molecule with Increased Proteolytic Stability
Biologics provide much higher specificity for an identified target than small molecules, resulting in low toxicity and limited side effects. Active pharmaceutical ingredients or chelating agents can be conjugated to polypeptides to achieve targeted delivery of these small molecules in therapeutic and diagnostic applications to reduce their general toxicity. For effective delivery, the intact conjugate must have sufficient time to accumulate in the target tissue. Half-life increases with increasing size but may be limited by proteolytic degradation and rapid filtration from the body before reaching the target tissue. To improve the circulating half-life large, hydrophilic, repetitive polymers, such as polyethylene glycol (PEG), have been conjugated to increase effective size and to hinder access by proteases. PEG has been used successfully, but it must be chemically conjugated to the product. To increase half-life but simplify production, a naturally protease-resistant polypeptide, XTEN, can be genetically fused to the protein of interest. A proof of concept study was conducted using a model fusion construct to investigate the ability of the claMP Tag to inhibit proteolysis. The effect of metal-bound to the claMP Tag on proteolysis was investigated and the sequence adjacent to the claMP Tag was varied to assess the effect of spacer residues on the rate of cleavage. The results from this proof of concept study were used to design a targeted imaging agent of cancers that are positive for epidermal growth factor receptor (EGFR). The native ligand EGF, which is rapidly cleared because of its small size, was engineered inline with XTEN and the metal-binding claMP Tag. Sites of degradation by serum proteases were identified and mutations are being explored to improve stability.