Fig. 1.

Affinity maturation of the Ang2-BD_RGD-based library bi-specific for α_vβ₃ integrin and Tie2-Fc. a Ang2-BD was presented on the yeast cell surface as a fusion with agglutinin proteins. Display levels were detected using primary antibodies against the C-terminal cMyc tag (chicken anti-cMyc antibodies) and phycoerythrin (PE)-conjugated anti-chicken antibodies. Binding to Tie2-Fc was determined using fluorescein isothiocyanate (FITC)-conjugated anti-human Fc antibodies. Binding to α_vβ₃ integrin was determined using FITC-labeled mouse anti-α_v integrin antibodies. b–g FACS analysis of the binding of the bi-specific Ang2-BD-based library to α_vβ₃ integrin in different screening steps. Quadrant gate statistics are indicated in each panel b negative control. c Ang2-BD_WT expression and α_vβ₃ integrin binding (10 nM). d Expression of the bi-specific Ang2-BD_RGD-based library and α_vβ₃ integrin binding (10 nM) at pre-sorting and e–g expression of the bi-specific Ang2-BD-based library and α_vβ₃ integrin binding (10 nM) after sorts 1, 3, and 5, respectively. h Binding of isolated yeast-displayed bi-specific Ang2-BD_RGD clones to Tie2 (20 nM). Data were normalized to the yeast surface expression levels of each clone and Tie2 binding of Ang2-BD_WT. i Binding of isolated yeast-displayed bi-specific Ang2-BD_RGD clones to α_vβ₃, α₅β₁, α_vβ₅, α₄β₇, α_IIbβ₃, and α₃β₁ integrins (50 nM). Data were normalized to the yeast surface expression level of each clone. Data shown represent the average of triplicates from independent experiments, and error bars represent the standard error of the mean