Dysregulation of cell signaling pathways that mediate proliferation, survival, and migration are an underlying cause of cancer, and result in invasion and metastasis of many human tumors. In particular, dysregulation and over-expression of the Met tyrosine kinase receptor correlates to poor prognosis in many human tumors, making it an attractive target for therapeutic intervention. There are currently no FDA-approved therapeutics targeting the Met receptor;however, a few candidate molecules have recently entered early stage clinical trials. Therefore, molecules that potently inhibit Met receptor activation would have a significant clinical impact on cancer therapy. In addition, studies to develop Met-targeted molecular imaging agents for non- invasive visualization of Met expression in vivo have been extremely limited. The availability of such imaging agents would aid in cancer diagnosis, staging, and disease management, as well as help identify patients who would be good candidates for Met-targeted therapies. To develop robust Met-targeting agents we used the N- terminal and first Kringle domain (NK1) of the natural Met activating ligand, hepatocyte growth factor (HGF), as a basis for engineering potent Met receptor antagonists. Using directed evolution, we engineered NK1 mutants with significant improvements in Met binding affinity and thermal stability compared to wild-type NK1. Rationally-designed, site-directed mutations introduced into these NK1 proteins transformed them into Met receptor antagonists.
In Aim 1 of the proposal, we will perform pre-clinical studies on fluorescently-labeled and radiolabeled NK1 mutants to test their ability to non-invasively image Met expression in living subjects, with the goal of developing them as in vivo molecular imaging agents.
In Aim 2, we will perform pre-clinical studies to measure the effects of NK1 mutants on tumor growth, metastasis, and angiogenesis in several mouse tumor models. During the course of treatment, non-invasive imaging will be used to monitor growth and progression of the primary tumor and metastases, and to monitor changes in Met expression and metabolism at the tumor site.
In Aim 3, we will fully characterize the binding of a larger panel of engineered NK1 mutants to Met-expressing tumor cells, and will determine their ability to dimerize and subsequently inhibit Met receptor activation.
In Aim 4, we will use these engineered NK1 proteins to probe sequence-structure-function relationships of ligand- receptor interactions in the Met receptor system, and provide biochemical and biophysical insight into their mechanism of action. In all four aims, results will be compared to wild-type NK1 to determine the effects of Met receptor binding affinity and protein stability on biological activity in cell culture and animal models. Upon completion of this proposal, we will have evaluated the potential of engineered NK1 proteins as molecular imaging and therapeutic agents in pre-clinical models, an important step on the path to clinical translation.
We developed high affinity, protein-based inhibitors of Met, a receptor that is over-expressed on many human cancers. These engineered Met-targeting proteins will be used to open up new research areas in cancer biology, cancer therapy, molecular imaging, and structure-based drug design. The studies we are proposing will also evaluate the potential of these engineered Met-targeting proteins for clinical translation as cancer diagnostics and therapeutics.
|Jones 2nd, Douglas S; Tsai, Ping-Chuan; Cochran, Jennifer R (2011) Engineering hepatocyte growth factor fragments with high stability and activity as Met receptor agonists and antagonists. Proc Natl Acad Sci U S A 108:13035-40|