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Utilizing combinatorial engineering to develop Tie2 targeting antagonistic angiopoetin-2 ligands as candidates for
Tomer Shlamkovich1, Lidan Aharon1, William A Barton2
1Department of Biotechnology Engineering, and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
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.
Insights
Researchers engineered novel angiopoietin (Ang)2-derived Tie2 antagonists using protein engineering. These potent inhibitors show promise for cancer therapy and angiogenesis research.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- The receptor tyrosine kinase (RTK) Tie2 is crucial for cancer cell proliferation, survival, migration, and angiogenesis.
- Targeting Tie2 offers significant therapeutic potential in cancer treatment.
- Existing Tie2 inhibitors have limited success, with no FDA-approved selective therapies available.
Purpose of the Study:
- To develop a new generation of angiopoietin (Ang)2-derived Tie2 antagonists.
- To create novel cancer therapeutics and tools for studying angiogenesis.
- To engineer high-affinity Tie2 antagonists using a protein engineering approach.
Main Methods:
- Utilized a combinatorial protein engineering approach with a yeast surface display (YSD) library.
- Designed an Ang2 binding domain (Ang2-BD) mutant library to prevent ligand multimerization and receptor activation.
- Screened the library using high-throughput flow cytometry to identify enhanced Ang2-BD variants.
Main Results:
- Selected Ang2-BD variants demonstrated high affinity for soluble and cellular Tie2.
- These variants potently inhibited Tie2 phosphorylation, endothelial capillary tube formation, and cell invasion.
- The study elucidated sequence-structure-function relationships and mechanisms of action for antagonistic Ang mutants.
Conclusions:
- Engineered Ang2-derived Tie2 antagonists represent a promising new class of cancer therapeutics.
- The protein engineering approach using natural ligands as scaffolds is applicable to other biomedical targets.
- These antagonists serve as valuable tools for investigating angiogenesis and Tie2 signaling pathways.