Figure 2. Characterization of zybody binding. (A) The affect of position of the MRD on Ang2 binding was measured for trastuzumab (TRA) and four bispecific, bi-valent trastuzumab-based zybodies containing an Ang2-binding MRD, a1. Zybodies were assessed for their ability to bind to Ang2 (left), ErbB2-Fc (center) and FcγRIIIa (right). (B) Simultaneous binding to cell surface ErbB2 and soluble Ang2 was assessed by FACS after incubating SKBR3 cells with (a) anti-human Igκ FITC, (b) TRA-a1H and anti-human Igκ FITC, (c) TRA-a1H and Ang2-biotin, (d) TRA-a1H, anti-human Igκ FITC and Ang2-biotin. Ang2-biotin binding was detected using streptavidin-PE.