Figure 4. A bispecific zybody enhances inhibition of IGF-induced binding, signaling and proliferation. (A) MCF-7 and MCF-7ErbB2 cells were treated with 63 nM of various combinations of trastuzumab (TRA) and bispecific zybodies that target IGF-1R (TRA-i1H or ADA-i1H). Twenty-four hours after treatment, the ability of cells to bind biotinylated IGF-1 was measured by FACS. (B) Cells treated as in (A), were further stimulated with IGF1 for 10 min and solubilized in lysis buffer. Immunoblotting indicates a reduction in IGF-1R levels and IGF1-induced phosphorylated Akt (pAkt) in MCF-7ErbB2 cells treated with TRA-i1H. Total Akt (tAkt) and β-tubulin levels were unchanged. (C) Inhibition of proliferation of MCF-7 and MCF-7ErbB2 cell was assayed after treatment for 96 h with the combinations described in (A).