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.

Keywords: angiogenesis; antagonistic activity; directed evolution; protein engineering; protein-protein interactions.

Figure 1. Screening of first- and second-generation Ang2-BD libraries against soluble Tie2

Shown is a FACS analysis of yeast expressing Ang2-BD. (A) Negative control. (B) Ang2-BD_WT expression and binding of Tie2 (10 nM). (C) Ang2-BD library expression and binding of Tie2 (10 nM). (D) Ang2-BD library expression and binding of Tie2 (10 nM) after five rounds of sorting. (E) Ang2-BD_C1.70 expression and binding of Tie2 (5 nM). (F) Ang2-BD_C1.70 library sort expression and binding of Tie2 (5 nM). (G–I) Ang2-BD_C1.70 library expression and binding of Tie2 (5 nM) after sorts 1, 3 and 5, respectively. Sorts 2–5 were conducted using gates similar to the one shown in panel F.

Figure 2. Localization of Ang2-BD mutations

(A) The Ang2/Tie2 complex (PDB 2GY7) is shown in cartoon format with Ang2 in green and Tie2 in blue. The location of mutations in Ang2-BD_C2.36 are shown as red spheres at the Cα atom and labelled accordingly. The panel on the left is rotated 90 degrees along the y-axis relative to the image on the right. (B) A close-up view of the Ang2/Tie2 binding interface highlighting the location of the six mutations. A calcium atom is shown in space filling format while the mutations are in ball-and-stick.

Figure 3. Binding of Ang2-BD variants to recombinant and cell-expressed human Tie2

(A) Representative SPR sensorgrams of binding of Ang2-BD variants to immobilized Tie2. The ranges of protein concentrations analyzed are indicated in parentheses: Ang2-BD_WT (18.75 nM – 300 nM); Ang2BD_C1.70 (6.25 nM – 100 nM); Ang2-BD_C2.36 (5 nM – 80 nM); Ang2-BD_C2.36 glycosylated (6.25 nM – 100 nM). (B) Binding of Ang2-BD variants to TIME cell line: 1 ×10⁵ cells were incubated with indicated proteins (Ang2-BD_WT, Ang2-BD_{C1.70 and} Ang2-BD_C2.36, red, green and blue, respectively) for 2 h at 4°C with a gentle agitation. Mean fluorescence values were determined by flow cytometry using a fluorescently labeled antibody against a FLAG epitope tag. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean. *indicates P value < 0.05 for comparison of results between Ang2-BD variants at the same concentration. (C) Competitive binding assay of Ang2-BD_WT, Ang2-BD_C1.70 and Ang2-BD_C2.36, without Ang1 (red, green and blue, respectively) and with 400 ng/ml Ang1 (pink, brown and yellow, respectively) and 1000 ng/ml Ang1 for Ang2-BD_C2.36 competition (grey). *indicates P value <0.05 for comparison of results between Ang2-BD variants with and without Ang1. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean.

Figure 4. Inhibition of Tie2 phosphorylation by Ang2-BD variants

(A) TIME cells were treated with control buffer (basal level, black), 200 ng/ml of Ang1 (green), 200 ng/ml of Ang1 and 1 μM Ang2-BD_WT (brown), 1 μM Ang2-BD_WT(red), 200 ng/ml of Ang1 and 1 μM Ang2-BD_c2.36 (blue) and 1 μM Ang2-BD_C2.36 (purple) for 15 min for Tie2 phosphorylation. (B) Cell lysates were analyzed by western blot using antibodies against pTie2, Tie2 and β-actin. # indicates P value < 0.05 for comparison of results between Ang2-BD_WT and Ang2-BD_C2.36; ## indicates P value < 0.01 for comparison of results between Ang1 + Ang2-BD_WT and Ang2-BD_WT and between Ang1 + Ang2-BD_C2.36 and Ang2-BD_C2.36; *indicates P value < 0.05 for comparison of results between Ang1 and Ang1 + Ang2-BD_WT; **indicates P value < 0.01 for comparison of results between Ang1 and Ang1 + Ang2-BD_C2.36. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean.

Figure 5. Inhibition of tube formation in endothelial cells by Ang2-BD variants

(A) TIME cells were treated with the indicated proteins. (B) Control buffer (cells only, black), 200 ng/ml of Ang1 (green), 2 μM, 4 μM and 8 μM Ang2-BD_WT (red, pink and brown respectively), 2 μM, 4 μM and 8 μM Ang2-BD_C2.36 (blue, yellow and grey respectively). Tube structures were analyzed for the number of generated junctions and the total tube length. *indicates P value < 0.05 for comparison of results between cells alone and tested proteins. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean. Scale bar, 500 μm.

Figure 6. Inhibition of endothelial cells invasiveness by Ang2-BD variants

(A) TIME cells were treated with indicated proteins in Boyden chambers. (B) Control buffer (cells only, black), 2 μM and 4 μM Ang2-BD_WT (red and pink, respectively), 2 μM and 4 μM Ang2-BD_C2.36 (blue and brown, respectively). The invading cells accumulated on the bottom of membrane were counted in 16 frames for each membrane and analyzed for the number of cells. *indicates P value < 0.05 for comparison of results between cells only and cells + tested proteins. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean. Scale bar, 200 μm.