Figure S5 from Improving Intracellular Delivery of an Antibody–Drug Conjugate Targeting Carcinoembryonic Antigen Increases Efficacy at Clinically Relevant Doses In Vivo

CEA Antibody Internalization Rate. LS174T cells were incubated continuously with 20 nM of antibody, and the internalization rate was measured as previously reported1. For both antibodies, total and surface fluorescence increased over the course of the experiment. The approximately tenfold difference in MFI between XAb and hMN-14 binding was due to the different flow cytometer voltage settings on the days the experiments were run(A). The increase in surface fluorescence is likely due to LS174T cell packing that can increase the time for binding equilibrium and delay surface fluorescence plateau. The main difference between XAb and hMN-14 conjugates was the differing internalization rates, consistent with literature reports2. The internalized fluorescence for XAb increased throughout the 8-hour incubation while hMN-14 internalized fluorescence tapered off towards the end of the continuous incubation. The data were used to determine the time integral of the surface fluorescence and plotted against the internal fluorescence (B). A linear fit was used to calculate the slope which corresponds to ke.