Utilizing combinatorial engineering to develop Tie2 targeting antagonistic angiopoetin-2 ligands as candidates for anti-angiogenesis therapy

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.

All Good Things Must End: Termination of Receptor Tyrosine Kinase Signal

“…For instance, a developed mutant of angiopoieitin-2 ligand retained the ability to bind the receptor Tie2 but negatively influenced the ligand multimerization and RTK dimerization and activation. The angiopoietin2-derived Tie2 antagonist strongly inhibited Tie2 phosphorylation, affected endothelial capillary tube formation, and influenced cell invasion [87].…”

Engineering Stem Cell Factor Ligands with Different c-Kit Agonistic Potencies

“…Current methodologies aimed at generating potent RTK antagonists and agonists are thus based on protein engineering designed to modify ligands by changing both their affinities [ 17 ] and their self-dimerization status [ 14 ]. However, since affinity and self-dimerization are not mutually exclusive, the engineering of optimized protein-based antagonists or agonists must find the delicate balance between improved binding affinity and impaired self-dimerization [ 18 ]. In practical terms, combinatorial site-directed engineering of both ligand-ligand and ligand-receptor interactions provides the means to generate improved therapeutic mediators and to gain insights into the dynamics of RTK-ligand interactions.…”

Residue‐level determinants of angiopoietin‐2 interactions with its receptor Tie2

“…Indeed, anti‐angiogenic therapies that center on the comparable RTK ligand VEGF are already in the clinic and more angiogenesis‐targeted drugs are in development . Different strategies have been explored to prevent Ang2 binding to Tie2 as potential therapies for ophthalmic diseases and for cancer . Nevertheless, the molecular determinants of Ang2 binding to Tie2 are not fully understood at the individual amino acid level, impeding advances toward a therapeutic goal of re‐engineering Ang2 into an effective antagonist.…”