Abstract
In many human cancers, the receptor tyrosine kinase (RTK) Tie2 plays important roles in mediating proliferation, survival, migration and angiogenesis. Thus, molecules that could potently inhibit activation of the Tie2 receptor would have a significant impact on cancer therapy. Nevertheless, attempts to develop Tie2-targeted inhibitors have met with little success, and there is currently no FDA-approved therapeutic selectively targeting Tie2. We used a combinatorial protein engineering approach to develop a new generation of angiopoietin (Ang)2-derived Tie2 antagonists as potential cancer therapeutics and as tools to study angiogenesis. The construct for designing a yeast surface display (YSD) library of potential antagonists was an Ang2 binding domain (Ang2-BD) that retains Tie2 binding ability but prevents ligand multimerization and receptor dimerization and activation. This mutant library was then screened by quantitative high-throughput flow cytometric sorting to identify Ang2-BD variants with increased expression, stability and affinity to Tie2. The selected variants were recombinantly expressed and showed high affinity to soluble and cellular Tie2 and strongly inhibited both Tie2 phosphorylation and endothelial capillary tube formation and cell invasion compared to the parental Ang2-BD. The significance of the study lies in the insight it provides into the sequence-structure-function relationships and mechanism of action of the antagonistic Ang mutants. The approach of using a natural protein ligand as a molecular scaffold for engineering high-affinity agents can be applied to other ligands to create functional protein antagonists against additional biomedical targets.
Highlights
The receptor tyrosine kinases (RTKs) Tie2 and Tie1 and their angiopoietin (Ang) endothelial growth factor ligands are known to be involved in the formation of blood vessels— both developmental and pathological [1,2,3,4,5,6]
Ang2 binding domain (Ang2-BD) was cloned into a yeast surface display (YSD) plasmid and presented on the yeast cell surface as a fusion to agglutinin proteins
We have shown that this method is an effective strategy for creating ligand-based RTK receptor inhibitors and molecular tools to study the effects of growth factor/RTK recognition on receptor activation and cell function
Summary
The receptor tyrosine kinases (RTKs) Tie and Tie and their angiopoietin (Ang) endothelial growth factor ligands (in the human, Ang1–Ang4) are known to be involved in the formation of blood vessels— both developmental and pathological [1,2,3,4,5,6]. It is known that Ang mediates endothelial cell growth, proliferation, and neovascularization through binding and activation of Tie2 [9]. Upon binding to Ang multimers, the Tie receptor oligomerizes, bringing its kinase domains into close proximity and thereby enabling the kinase domains to phosphorylate each other to promote cell proliferation, migration, and sprouting [14]. Activation of Tie via tyrosine phosphorylation www.impactjournals.com/oncotarget initiates the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, which leads to Akt and MAPK/ERK signaling and to both endothelial cell survival and migration phenotypes [15, 16]
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