Interestingly, MCF-7 cells were generally more sensitive to the ADCs at higher concentrations, consistent with the trend observed for the unconjugated azides (Figure ). Specifically, we observed a 6-fold and 2-fold difference in cytotoxicity between the two cell lines for conjugates 5 and 7, respectively (Figure ). While the DpC-based ADC 6 was largely non-cytotoxic to both cell lines, the linker positioning played a crucial role in MCF-7 cells, as 8 reduced cell viability by 8fold. On the other hand, ortho positioning of the linker was more favorable for the Dp44mT-based ADCs as both cell lines were relatively less susceptible to conjugate 7 compared to 5. Overall, these findings suggest that the conjugated DpT payload exerts a greater influence on the in vitro activity, overriding the a priori expected selectivity offered by trastuzumab. Furthermore, while linker position did not affect the cytotoxicity of the azides, ADCs displayed significant potency differences depending on the linker position.