(47e) Engineered Hepatocyte Growth Factor Mutants: New Tools for Tissue Regeneration and Vascularization

Hepatocyte growth factor (HGF), through activation of its tyrosine kinase receptor c-MET, has shown great promise in regenerative medicine; however, the instability and poor expression yield of recombinant HGF have been a severe bottleneck to its clinical translation. Using rational and combinatorial methods, we engineered a protein fragment composed of the N terminal domain and first Kringle domain of HGF, termed NK1. Compared to full-length HGF (~105 kDa), the engineered NK1 fragment (termed eNK1) is much smaller (~20 kDa), has increased thermal stability by more than 15°C, and can be recombinantly expressed at an order of magnitude higher yield in yeast than native NK1. Dimerization of HGF’s NK1 domains is a prerequisite to c-MET dimerization and activation. Thus, to develop a more potent agonist, we created a dimeric eNK1 protein via covalent crosslinking through an N-terminal disulfide bond. Biophysical and biological characterization of the dimeric eNK1 protein will be discussed, including affinity to c-MET, downstream phosphorylation, endothelial migration, and vascularization. Remarkably, we found that the eNK1 dimers elicited significantly greater agonistic activity than the eNK1 monomer, approaching activity levels of full-length HGF. Our ultimate goal is to develop these engineered proteins as novel therapeutics for promoting tissue regeneration. Funded by NIH NCI R01CA151706.