## 2 Results and Discussion
This binding increased with HER2 expression as mean fluorescence intensity varied from 14.2 in MCF-7 cells expressing very low levels of HER2, to \(\sim\)84 in T-47D and SK-BR-3 cells expressing intermediate levels of HER2 and \(\sim\)180 in SK-OV-3 cells expressing very high level of the receptor, respectively (Figure 2, Table 1). Interestingly, similar results were observed for trastuzumab alone, indicating that grafting of the DNA mimic with the caproic linker did not affect the binding capacity of the immunoconjugate. We then investigated whether the ADC could be internalized into cells. For this purpose, cells were treated with a fixed concentration of trastuzumab or of the ADC (15 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) for 2 h at 4 \({}^{\circ}\)C or at 37 \({}^{\circ}\)C, and immunofluorescence analyses were performed (Figure 3 and Figure S4). The results of Figure 3 showed that, in SK-BR-3 and SK-OV-3 cells expressing the highest membrane levels of HER2, fluorescence was strictly localized at the cell surface whereas small intracellular patches of fluorescence could be detected when cells were treated at 37\({}^{\circ}\)C, which indicated an internalization of the ADC. Similar results were obtained for trastuzumab used as a positive control (Figure S4), in accordance with previous studies [40]. Internalization of both the ADC and trastuzumab was also observed in T-47D cells with a comparable change in fluorescence patches distribution between 4 \({}^{\circ}\)C and 37 \({}^{\circ}\)C, however, could not be evidenced in MCF-7 cells due to the very low level of expression of HER2 (Figure S4). We then evaluated the effect of the ADC on the growth of breast and ovarian cancer cells using the standard sulforhodamine B in vitro assay (Figure 4). This effect was compared to the effect of equimolar concentrations of trastuzumab or of the (Q\({}^{\mathrm{pho}}\))\({}_{16}\) DNA mimic alone, taking into account that the drug-to-antibody ratio was approximately three. The results showed that the DNA mimic alone did not have any effect on cell growth, which is in accordance with our previous observations [15] and confirms the difficulty of delivering these polyanionic moieties into cells without the use of a transfection agent. Figure 3: Internalization of the trastuzumab-DNA mimic conjugate in HER2 cells expressing HER2 at 4 \({}^{\circ}\)C and 37 \({}^{\circ}\)C as evaluated by immunofluorescence. Exponentially growing cells were incubated with 15 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of trastuzumab (TZ) or the trastuzumab-DNA mimic conjugate (ADC) for 2 h at 4 \({}^{\circ}\)C or 37 \({}^{\circ}\)C and were fixed. Localization of the antibodies was revealed by immunofluorescence using an FITC labeled antibody as described in the Materials and Methods section. Untreated cells were used as a negative control. They also suggested that DNA mimics cannot exert their cytotoxic effect via an interaction with the cell surface. We found that, in our conditions, trastuzumab had only a minor effect on cell growth, even at the highest concentration used (100 ug/mL), with less than 20% growth inhibition, which is consistent with a previous study reporting the in vitro effects of this monoclonal antibody in the same models [41]. When compared to trastuzumab, our conjugate was more efficient at inhibiting cell growth only for the highest concentration of the drug used, i.e., 100 ug/mL (Figure 4). IC\({}_{50}\)s of our conjugate could only be obtained in SK-OV-3 and SK-BR-3 cells overexpressing HER2 (~50 ug/mL and ~100 ug/mL, respectively). Of note, 100 ug/mL of the trastuzumab-DNA mimic conjugate corresponds to a foldamer concentration of approximately 1.8 uM, which is in the same range of the concentrations that were transfected to obtain the maximal growth inhibition in HCT116 and HEK293 human cancer cell lines [15]. Other trastuzumab-drug conjugates were synthesized using highly cytotoxic agents as payloads, and were shown to be much more potent than trastuzumab in vitro and in vivo, even in trastuzumab-resistant models [41].