## 2 Results and Discussion
Repeated ultrafiltration and filtration yielded the purified ADC **4** with an average DAR of 3.0 (for MS analysis and DAR calculation: see Table S1 and Figure S5). The successful conjugation of the DNA mimic onto trastuzumab demonstrated that it is possible to graft high molecular weight payloads to therapeutic antibodies. Previously, NAs conjugation onto antibodies (IgG or fragments) has been described for siRNA, allowing a rapid clearance during plasma circulation and a specific uptake of siRNA in cancer cells [34; 35]. Conversely to classical hydrophobic payloads, both siRNA and foldamers have the advantage of exhibiting hydrophilic properties, allowing better solubility in water and a bioconjugation process free of organic solvent. Figure 1: (**A**) Schematic representation of a site-specific ADC comprising the anti-HER2 trastuzumab conjugated to a polyanionic foldamer via a diphenylthio-maleimido-caproic acid linker (diphenylthio-Mal-Cap) with a DAR of 3 (drug-to-antibody ratio, corresponding to the number of foldamers grafted onto the mAb). (**B**) Coupling reaction performed on solid phase between the protected foldamer **1** and the diphenylthio-Mal-Cap linker **2** to produce the diphenylthio-Mal-Cap-foldamer polyanionic **3** after resin cleavage and side chain deprotection. (**C**) The ADC **4** was obtained by complete reduction (TCEP) of all four interchain disulfide bridges followed by a site-specific bioconjugation reaction via a diphenylthio-Mal-Cap linker **2**. During the foldamer solid phase synthesis and the coupling reaction shown in (**B**), the phosphate groups of the quinoline monomers are protected as _tert_-butyl esters. TCEP = tris(2-carboxyethyl)phosphine. We then characterized the biological activity of the newly synthesized ADC using three breast (MCF-7, T-47D and SK-BR-3) and one ovarian (SK-OV-3) cancer cell lines expressing various levels of HER2 [36; 37; 38; 39]. We first wanted to validate whether the ADC could recognize its target at the cell surface, i.e., to assess whether the large payload (MW 4907 Da) alters in any way the HER2-trastuzumab interaction. We thus evaluated ADC binding to the receptor by FACS (Figure 2). Binding of the ADC was compared to that of trastuzumab alone or of the irrelevant antibody 13R4 used as a negative control. While no binding could be evidenced for the 13R4 antibody with a mean fluorescence intensity value ranging from 0.3 to 0.6 (Table 1), a significant binding could be observed for the ADC in all the cell lines tested. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **13R4** & **TZ** & **ADC** \\ \hline \multirow{2}{*}{**MCF-7**} & **0.45** & **13.1** & **6.7** \\  & (0.6, 0.3) & (14.1, 12.2) & (7.2, 6.2) \\ \hline \multirow{2}{*}{**T-47D**} & **0.4** & **85.0** & **83.6** \\  & (0.3, 0.5) & (65.9, 104.0) & (65.1, 102.0) \\ \hline \multirow{2}{*}{**SK-BR-3**} & **0.4** & **88.0** & **84.6** \\  & (0.3, 0.45) & (89.0, 87.0) & (86.1, 83.0) \\ \hline \multirow{2}{*}{**SK-OV-3**} & **0.5** & **223.5** & **180.0** \\  & (0.5, 0.5) & (222.0, 225.0) & (177.0, 183.0) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Mean fluorescence intensity (Gmean) values following cell incubation with the ADC, trastuzumab or the irrelevant 13R4 antibody as measured by FACS. Values are the mean of fluorescence intensities obtained from two independent experiments (indicated in parentheses). Figure 2: Binding of the trastuzumab-DNA mimic conjugate to breast (MCF-7, T-47D, SK-BR-3) and ovarian (SK-OV-3) cancer cells expressing various levels of HER2 was evaluated by FACS. Cells were incubated with 10 \(\mu\)g/mL of the trastuzumab-DNA mimic conjugate (ADC), trastuzumab (TZ) or the irrelevant 13R4 antibody for 1 h. Then, cells were washed with cold PBS, trypsinized and \(1\times 10^{6}\) cells were analyzed by FACS. Untreated cells were used as a control.