Engineering Potent Met Receptor Agonists and Antagonists Based on the NK1 Fragment of Hepatocyte Growth Factor

The Met tyrosine kinase receptor is a critical cancer target that is overexpressed on many solid tumors, and molecules that inhibit Met activation hold significant promise for cancer therapy. Activation of Met by its ligand, hepatocyte growth factor (HGF), results in a range of cellular responses including cell motility, mitosis, angiogenesis, tubule morphogenesis, and resistance to apoptosis. Thus, there is also great interest in Met receptor agonists to promote wound healing and tissue repair. HGF is an 80 kDa protein composed of multiple domains that interact with and activate the receptor through heparin-mediated homodimerization. The minimal fragment of HGF that can activate Met is composed of its N-terminal domain and first Kringle domain (termed NK1). However, NK1 is a weak agonist, is unstable, and is difficult to express recombinantly, which has severely limited its therapeutic potential. Here, we used directed evolution to engineer NK1 mutants with substantial improvements in stability and recombinant expression yield compared to wild-type NK1. In addition, we used NK1 as a platform for creating potent Met receptor agonists and antagonists for regenerative medicine and cancer therapy applications, respectively.

First, using yeast surface display, we evolved mutants of the NK1 fragments with marked improvements in stability. Engineered NK1 mutants exhibited significant improvements in thermal stability (T_m~60-70 °C) compared to wild-type NK1 (T_m=51 °C). A linear relationship was observed between yeast surface expression levels and thermal stability. A similar correlation was observed for yeast surface expression levels and recombinant expression yield, with the highest expressing mutants purified at levels over 30 mg/L in the yeast strain Pichia pastoris. The NK1 variants isolated from our library screens contained a mutation at position 127, which has been shown to mediate ligand homodimerization, and as a result did not activate Met.  Point mutants that further disrupted this dimerization interface functioned as Met antagonists by inhibiting HGF-mediated cell scatter and uPA activation. We are currently testing the ability of these Met receptor antagonists to inhibit tumor growth and metastasis, and the potential of labeled NK1 proteins for use as non-invasive molecular imaging agents in living subjects.

Building on this mechanistic understanding of the NK1-Met interaction, we created covalent NK1 homodimers to function as Met agonists. Remarkably, the NK1 dimers exhibited an order of magnitude greater agonistic activity compared to wild-type NK1, with activity levels approaching that of full-length HGF. However, the engineered NK1 dimers are comprised of subunits with substantially improved stability and can be recombinantly expressed at high yield in yeast, compared to HGF, which must be solubly produced in mammalian cell culture. These NK1 agonists will open up new research areas in regenerative medicine where HGF has shown great promise in liver, renal, and cardiac tissue engineering but currently must be introduced by gene transfer or viral delivery methods due to HGF instability and inability to generate adequate amounts of recombinant protein.

Funding: NIH NCI R21CA131706, NIH NCI R01CA151706, Siebel Scholars Program