Measuring HER2 Binding Affinity Assay

The binding affinity of [¹²⁵I]IB-Mal-d-GDDDK-5F7 and [¹²⁵I]IB-Mal-d-GEEEK-5F7 for HER2 was determined using a saturation binding assay on the HER2-expressing BT474M1 cell line. Cells were seeded in 24-well plates at a density of 8 × 10⁴ cells/well and incubated at 37 °C for 24 h. On the next day, the cells were incubated in triplicate with increasing concentrations (0.1–100 nM; final volume 600 μL) of [¹²⁵I]IB-Mal-d-GDDDK-5F7 or [¹²⁵I]IB-Mal-d-GEEEK-5F7 for 2 h at 4 °C. A parallel assay was performed in which cells were coincubated with a 100-fold molar excess of trastuzumab, which competes with 5F7 for HER2 binding [17 (link)], to determine nonspecific binding at each concentration. The medium containing unbound activity was removed, and cells were washed twice with cold PBS and solubilized by treatment with 1 M NaOH (0.5 mL) at 37 °C for 10 min. Cell-associated activity was determined using a Perkin Elmer Wizard II (Shelton, CT, USA) dual-channel gamma counter. The dissociation constant (K_D) was determined by nonlinear regression using GraphPad Prism software (version 5.01). Triplicate samples were used for each concentration, and the entire experiment was repeated twice.