Pharmacokinetic study

Female, 6–8 weeks old CD-1® IGS mice (Charles River Laboratories, Strain Code: 022) were randomly assigned to each group (n = 3 for MMAE DAR 4 ADC; n = 4 for other groups) and were administered intravenously with the unmodified N297A anti-HER2 mAb or each ADC at a dose of 3 mg kg⁻¹. Blood samples (5 µL) were collected from each animal via the tail vein at each time point (15 min, 5 h, 1 day, 2 days, 4 days, 9 days, and 14 days) and diluted in 495 µL of 5 mM EDTA in PBS. After removal of cells by centrifugation (10 min at 9400 × g at 4 °C), plasma samples were stored at −80 °C until used for subsequent sandwich ELISA. For determination of the total antibody concentration (both conjugated and unconjugated), a high-binding 96-well plate (Corning) was coated with goat anti-human IgG Fc antibody (500 ng per well) diluted in 100 mM sodium carbonate buffer (pH 9.4). After overnight incubation at 4 °C, the plate was blocked with 100 µL of 2% BSA in PBS containing 0.05% Tween 20 (PBS-T) at room temperature for 1 h. Subsequently, the solution was removed and each diluted plasma sample (100 µL in PBS-T containing 1% BSA) was added to each well, and the plate was incubated at room temperature for 2 h. After each well was washed three times with PBS-T, 100 µL of goat anti-human IgG Fab-HRP conjugate (1 : 5000) was added. After being incubated at room temperature for 1 h, the plate was washed and color development was performed as described above (see the section of “Cell-based ELISA assay”). For determination of ADC concentration (conjugated only), assays were performed in the same manner using human HER2 (100 ng per well, ACROBiosystems) for plate coating, mouse anti-MMAE/F antibody (1 : 5000), and goat anti-mouse IgG-HRP conjugate (1 : 10,000) as secondary and tertiary detection antibodies, respectively. All assays were performed in triplicate. Concentrations were calculated based on a standard curve. Half-life of each conjugate at the elimination phase (t_1/2β) was estimated using methods for non-compartmental analysis^54,55. PKSolver (a freely available menu-driven add-in program for Microsoft Excel)^54,55 was used to calculate t_1/2β and area under the curve (AUC_0–∞, h × μg mL⁻¹).