Intrigued by the findings, we picked ADC Tras-(Dp44mT-O)2 5, which displayed the highest potency in both cell lines, to perform further experiments. Trastuzumab and its ADCs exert their anticancer activity in a stepwise manner, initially by binding to the extracellular domain of the HER2 receptor, followed by internalization of the payload via endocytosis. We hypothesized that the sustained presence of the conjugates during the entire incubation period might be negatively affecting trastuzumab's selectivity for SK-BR-3 cells. To test this, we incubated compound 5 with both cell lines for 2 h, swapped for fresh media, and continued the incubation for an additional 118 hours (Figure ). Interestingly, this adjustment did not alter the cytotoxicity profile, as 5 remained more toxic to MCF-7 and failed to minimize the viability of SK-BR-3 cells even at the highest concentrations, underscoring that the observed activity originates from the toxicity of the payload. In accordance with these results, the control Dp44mT's activity also showed a significant reduction in SK-BR-3 cells (Figure ). Additionally, ADCC was observed for Tras at higher concentrations against SK-BR-3 cells, a pattern that was also evident for 5, implying that Tras-dependent activity persisted. To further ascertain Dp44mT's prominent role in the activity of the ADCs, we conjugated 1 to cetuximab, a mAb that binds the HER1 receptor. As illustrated in Figure , the unconjugated control antibody Cetux proved non-cytotoxic to both cell lines. Consistent with previous findings, cetuximab conjugate 9 demonstrated a greater cytotoxic effect against MCF-7 cells than SK-BR-3 cells, where it failed to achieve a 100% decrease in cell viability even at the highest concentrations. Notably, we did not observe the Tras-like ADCC against SK-BR-3 cells with Cet, further validating the selectivity of Tras towards the cell line.