Human breast cancer cells in passage 4 or 5 or U937 cells in passage 3 were seeded in 96-well culture plates at a density of 15,000 cells/well in 90 uL culture media. Cells were then treated in duplicates with anti-HER2 ARC-FDCs, anti-HER2 Fab, anti-HER2 Fab-CD38 fusions, or MMAF-alkyne that were prepared in 3-fold serial dilution starting from 300 nM. Cells treated with 5 uM paclitaxel (MilliporeSigma, MA) and PBS were included for 0% and 100% viability controls, respectively. Following incubation at 37°C in an incubator with 5% CO2 for 72 h, cells were added with MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reagent (Thermo Fisher Scientific, MA), incubated for 3 h, and lysed with 20% SDS in 50% dimethylformamide. Absorbance at 580 nm was then measured with a Synergy H1 plate reader after 1-hour incubation at 37°C with 5% CO2. EC50 was calculated by the three-parameter nonlinear regression fitting in GraphPad Prism Version 9.4.0 (GraphPad software, CA).

Human breast cancer cells in passage 4 or 5 or U937 cells in passage 3 were seeded in 96-well culture plates at a density of 15,000 cells/well in 90 uL culture media. Cells were then treated in duplicates with anti-HER2 ARC-FDCs, anti-HER2 Fab, anti-HER2 Fab-CD38 fusions, or MMAF-alkyne that were prepared in 3-fold serial dilution starting from 300 nM. Cells treated with 5 uM paclitaxel (MilliporeSigma, MA) and PBS were included for 0% and 100% viability controls, respectively. Following incubation at 37°C in an incubator with 5% CO2 for 72 h, cells were added with MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reagent (Thermo Fisher Scientific, MA), incubated for 3 h, and lysed with 20% SDS in 50% dimethylformamide. Absorbance at 580 nm was then measured with a Synergy H1 plate reader after 1-hour incubation at 37°C with 5% CO2. EC50 was calculated by the three-parameter nonlinear regression fitting in GraphPad Prism Version 9.4.0 (GraphPad software, CA).

Human breast cancer cells in passage 4 or 5 or U937 cells in passage 3 were seeded in 96-well culture plates at a density of 15,000 cells/well in 90 uL culture media. Cells were then treated in duplicates with anti-HER2 ARC-FDCs, anti-HER2 Fab, anti-HER2 Fab-CD38 fusions, or MMAF-alkyne that were prepared in 3-fold serial dilution starting from 300 nM. Cells treated with 5 uM paclitaxel (MilliporeSigma, MA) and PBS were included for 0% and 100% viability controls, respectively. Following incubation at 37°C in an incubator with 5% CO2 for 72 h, cells were added with MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reagent (Thermo Fisher Scientific, MA), incubated for 3 h, and lysed with 20% SDS in 50% dimethylformamide. Absorbance at 580 nm was then measured with a Synergy H1 plate reader after 1-hour incubation at 37°C with 5% CO2. EC50 was calculated by the three-parameter nonlinear regression fitting in GraphPad Prism Version 9.4.0 (GraphPad software, CA).

Human breast cancer cells in passage 4 or 5 or U937 cells in passage 3 were seeded in 96-well culture plates at a density of 15,000 cells/well in 90 uL culture media. Cells were then treated in duplicates with anti-HER2 ARC-FDCs, anti-HER2 Fab, anti-HER2 Fab-CD38 fusions, or MMAF-alkyne that were prepared in 3-fold serial dilution starting from 300 nM. Cells treated with 5 uM paclitaxel (MilliporeSigma, MA) and PBS were included for 0% and 100% viability controls, respectively. Following incubation at 37°C in an incubator with 5% CO2 for 72 h, cells were added with MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reagent (Thermo Fisher Scientific, MA), incubated for 3 h, and lysed with 20% SDS in 50% dimethylformamide. Absorbance at 580 nm was then measured with a Synergy H1 plate reader after 1-hour incubation at 37°C with 5% CO2. EC50 was calculated by the three-parameter nonlinear regression fitting in GraphPad Prism Version 9.4.0 (GraphPad software, CA).