Engineering of Therapeutic Proteins: Applications to Cancer and Fribrodysplasia Ossificans Progressiva

Abstract

The Notch signaling pathway has been implicated in many diseases due to deregulation in its role in many cellular processes. One major area of concern is its hand in cell proliferation of tumors in cancerous tissues. The extracellular Delta-like Ligand 1 (DLL1) that binds to Notch receptors has become a focus in recent studies in mice models by the Dikov Laboratory and their results suggest that a soluble cluster of a multivalent DLL1 may have therapeutic usage to suppress tumor growth. However, these DLL1 clusters are heterogeneous collages that are not suitable for in vivo testing or therapeutic drug creation. As a result, the Magliery Laboratory is expressing a variety of homogeneous DLL proteins. Through optimizing different expression, purification, and refolding techniques, numerous refolded DLL1 proteins have already been synthesized. These include a long ligand with three Epidermal Growth Factor (EGF) domains, a shortened ligand two EGF repeated domains, ligands with and without the N-terminal MNNL domain, and ligands with and without a C-terminal cysteine for PEGylation. A polyvalent structure of DLL1 ideally would have the potential to prevent tumor proliferation and cause apoptosis in cancerous tissues. Currently, we are optimizing the crosslinking of these fragments with PEG based linkers to make homogeneous, polyvalent constructs. Also, in vitro binding studies of these various proteins to the Notch receptor are underway in order to confirm the importance of each domain from our findings in the in vivo assays by the Dikov Laboratory. Fibrodysplasia Ossificans Progressiva (FOP) is a progressive heterotopic endochondral ossification (HEO) that results in bone formation in atypical extra-skeletal locations. A non-lethal heterozygous mutation in the activin receptor IA/activin-like kinase-2 (AVCR1/ALK2) was identified as a potential therapeutic target as this mutation exists in all cases of FOP. This complex is affiliated with bone morphogenetic protein (BMP) signaling receptor complexes, which is known to play a major role in bone and cartilage formation among other functions. Since the over-activity of the BMP signaling pathway is attributed as the cause to this disease, a possible inhibitor for excessive bone growth is a protein called Noggin, which binds and sequesters BMPs. Also a region of CV-2 called VWC has been found to bind to a different site on BMPs and sequester BMPs. In order to execute a number of different binding studies, optimizing the expression and purification of AVCR, BMP2, BMP4, Noggin, and VWC has been the initial goals of the Magliery Lab.