Engineering the Met receptor: a potent antagonist of tumor growth and metastasis

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DESCRIPTION (provided by applicant): The Met receptor tyrosine kinase is critical for mediating cell proliferation, survival and migration. The tendency for human tumors to invade and metastasize has been tied to the dysregulation of Met; therefore, Met is an extremely attractive target for therapeutic intervention. Interestingly, soluble Met extracellular domain (Met-ECD) acts as a potent antagonist of Met activation. Unfortunately, current methods for recombinant expression of Met-ECD have low yield, preventing both the full characterization of its biology and its deployment as a potential therapeutic. Our lab has robust and proven technology for the evolution, optimization, and expression of such challenging proteins, and Met-ECD is an ideal candidate for applying this technology. This work will accelerate biophysical characterization of Met ligand/receptor interactions, and applications of the Met receptor in cancer therapy and diagnostics. Aim 1: Engineer the full-length Met extracellular domain for high soluble expression levels in yeast. Directed evolution using yeast surface display provides us with a robust combinatorial platform to identify Met- ECD mutants that 1) possess the native receptor fold and 2) exhibit high soluble expression levels (mg/L) in yeast. Aim 2: Measure the ligand binding affinity and biological activity mediated by Met-ECD mutants. We will test the Met-ECD mutants isolated in Aim 1 to verify that engineered receptors with improved expression retain native ligand binding properties and biological function. We will fuse Met-ECD mutants to wild-type Met transmembrane and intracellular domains, and transfect these constructs into a mammalian cell line that expresses low levels of endogenous Met. We will test these cells lines for their ability to bind HGF ligand and induce cell signaling, comparing them to cells transfected with wild-type Met. These efforts will demonstrate the biological functions of Met-ECD mutants, validating them for future biophysical studies. Aim 3: Determine the ability of soluble Met-ECD mutants to bind to tumor cells and inhibit signaling. We will determine if soluble Met-ECD mutants function as receptor antagonists by measuring their ability to 1) bind to Met-expressing tumor cells and 2) inhibit ligand-dependent and ligand-independent activation of the Met kinase domain and downstream MAPK and PI3K signaling pathways. This Aim will validate Met-ECD mutants as receptor antagonists in a variety of cell types and motivate future research into their therapeutic potential. Project Narrative The availability of functional recombinant Met-ECD will profoundly impact future research into mechanisms of tumorigenesis, bacterial pathogenesis, and embryonic development, and will provide insight into how these processes can be manipulated. The fact that the Met-ECD itself could also be developed for applications in molecular imaging or cancer therapy is another example of this project's high impact.