Supplementary File from Improved Tumor Penetration and Single-Cell Targeting of Antibody–Drug Conjugates Increases Anticancer Efficacy and Host Survival

The supplementary data file includes additional immunofluorescence microscopy images showing the tumor distribution of T-DM1-680 alone, T-DM1-680 with unlabeled trastuzumab, and a higher dose of T-DM1-680. Supplemental in vitro assays include: single-agent trastuzumab cytotoxicity, cytotoxicity and quantitative uptake with constant total antibody concentration but different T-DM1 concentration, ADC cellular binding affinity with and without fluorophores, ADC cellular toxicity with and without fluorophores, and quantitative cellular uptake and degradation of dually labeled T-DM1. Supplemental in vivo data include: Kaplan-Meier curves with confidence intervals for each dosing level, individual tumor growth curves for each animal, mouse weight change during treatment, and plasma clearance of fluorescently labeled trastuzumab. The file also contains supplementary methods for immunofluorescence histology and surface to volume image analysis, and a supplementary discussion on the importance of bystander effects.