<p>Figure S1 - Ab033 EGFR binding kinetics. Figure S2 - Number of surface EGF receptors. Figure S3 - Workflow for internalization rate determination. Figure S4 - Comparison of Ab033 mAb and ADC internalization rates. Figure S5 - Visualization of internalization in H441 cells. Figure S6 - Visualization of full internalization time course in A431 cells. Figure S7 - Surface localization and internalization scores by imaging flow cytometry. Figure S8 - Quenching of AlexaFluor 488 fluorescence. Figure S9 - Colocalization of Ab033 to the lysosome. Figure S10 - Uptake and trafficking of Ab033 with pH sensitive dye. Figure S11 - Preparation of Ab033-E2/F2. Figure S12 - Drug concentration in media. Figure S13 - Mechanistic cellular model. Table S1 - Model equations. Table S2 - Model parameters.</p>
ARTICLE ABSTRACT
Antibody–drug conjugates (ADC) offer an avenue for specific drug delivery to target cells. Here, parameters with important roles in the cellular processing of ADCs were quantitatively measured for Ab033, an antibody against EGFR. In EGFR-overexpressing cancer cell lines, Ab033 internalized at rates of 0.047/min and 0.15/min for A431 and H441 cells, respectively. Once internalized, Ab033 either trafficked to the lysosome or was recycled; up to 45% of internalized Ab033 returned to the cell surface. Despite such recycling, intracellular accumulation of Ab033 continually increased over 24 hours. Ab033 was conjugated to form a dual toxin ADC containing both cleavable and non-cleavable linker-drug payloads for release rate comparisons. Intracellular concentrations of freed drug from cleavable linker were greater than from non-cleavable linker and exceeded 5 × 106 drug molecules per A431 cell after 24 hours. Compared with intracellular antibody accumulation, formation of released drug was delayed, likely due to the time needed for endo-lysosomal trafficking and subsequent linker/antibody proteolysis. Informed by the quantitative data, a cellular ADC model was constructed and used to summarize processing inefficiencies. Modeling simulations were conducted to determine parameter sensitivity on intracellular drug concentrations, with rates of EGFR internalization and recycling as well as ADC trafficking found to be the most sensitive toward final intracellular drug concentrations. Overall, this study shows Ab033 ADCs to be a viable strategy for delivery of cytotoxic drugs into tumor cells with subsequent modeling efforts able to highlight key processing steps to be improved for increased drug delivery. Mol Cancer Ther; 17(6); 1341–51. ©2018 AACR.
