In vitro cytotoxicity of CovADC-DM1 and CovADC-MMAE was compared to T-DM1 (Kadcyla, Roche, France) on BT-474 and SKBR3 Her2-positive breast cancer cell lines, MDA-MB-231 Her2-negative cell line was included in the assay to confirm target selectivity. All cell lines were obtained from ATCC and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin/streptomycin and 2 mM l-glutamine (Life technologies, France). Cells were maintained at 37 °C with 5% CO2 atmosphere. Optimal cell density of each cell lines was determined by IncuCyte S3 live-cell analysis system (Essence BioScience, UK). Cells were plated (SKBR3 and BT-474: 5000 cells per well, MDA-MB-231: 1,250 cells per well) on 96-well plates. Following the incubation for 24 h, cell culture medium was removed and serial dilutions of ADCs and free unconjugated drugs were added in triplicates. After 3 days incubation cytotoxicity was evaluated by sulforhodamine B assay (SRB assay kit, Sigma, France) based on the measurement of cellular protein content according to the manufacturer's instructions. Briefly, cells were fixed with 50% (wt/vol) TriChloroacetic Acid (TCA) at 4 °C (30 ul/well, final concentration 11.5%) for 1 h. After washing with dH2O cells were stained with 100 ul of 0.04% (wt/vol) SRB solution for 30 min and subsequently washed with 1% (vol/vol) acetic acid to remove unbound stain. Cellular protein bound dye was solubilized in 200 ul of 10 mM Tris base solution (pH 10.5) and the absorption was measured at 510 nm (Microplate reader, Tecan, Switzerland). The raw data was processed using GraphPad Prism 7.0 (GraphPad Software, San Diego, USA). Error bars in graphs represent SEM of two measurements.

In vitro cytotoxicity of CovADC-DM1 and CovADC-MMAE was compared to T-DM1 (Kadcyla, Roche, France) on BT-474 and SKBR3 Her2-positive breast cancer cell lines, MDA-MB-231 Her2-negative cell line was included in the assay to confirm target selectivity. All cell lines were obtained from ATCC and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin/streptomycin and 2 mM l-glutamine (Life technologies, France). Cells were maintained at 37 °C with 5% CO2 atmosphere. Optimal cell density of each cell lines was determined by IncuCyte S3 live-cell analysis system (Essence BioScience, UK). Cells were plated (SKBR3 and BT-474: 5000 cells per well, MDA-MB-231: 1,250 cells per well) on 96-well plates. Following the incubation for 24 h, cell culture medium was removed and serial dilutions of ADCs and free unconjugated drugs were added in triplicates. After 3 days incubation cytotoxicity was evaluated by sulforhodamine B assay (SRB assay kit, Sigma, France) based on the measurement of cellular protein content according to the manufacturer's instructions. Briefly, cells were fixed with 50% (wt/vol) TriChloroacetic Acid (TCA) at 4 °C (30 ul/well, final concentration 11.5%) for 1 h. After washing with dH2O cells were stained with 100 ul of 0.04% (wt/vol) SRB solution for 30 min and subsequently washed with 1% (vol/vol) acetic acid to remove unbound stain. Cellular protein bound dye was solubilized in 200 ul of 10 mM Tris base solution (pH 10.5) and the absorption was measured at 510 nm (Microplate reader, Tecan, Switzerland). The raw data was processed using GraphPad Prism 7.0 (GraphPad Software, San Diego, USA). Error bars in graphs represent SEM of two measurements.

In vitro cytotoxicity of CovADC-DM1 and CovADC-MMAE was compared to T-DM1 (Kadcyla, Roche, France) on BT-474 and SKBR3 Her2-positive breast cancer cell lines, MDA-MB-231 Her2-negative cell line was included in the assay to confirm target selectivity. All cell lines were obtained from ATCC and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin/streptomycin and 2 mM l-glutamine (Life technologies, France). Cells were maintained at 37 °C with 5% CO2 atmosphere. Optimal cell density of each cell lines was determined by IncuCyte S3 live-cell analysis system (Essence BioScience, UK). Cells were plated (SKBR3 and BT-474: 5000 cells per well, MDA-MB-231: 1,250 cells per well) on 96-well plates. Following the incubation for 24 h, cell culture medium was removed and serial dilutions of ADCs and free unconjugated drugs were added in triplicates. After 3 days incubation cytotoxicity was evaluated by sulforhodamine B assay (SRB assay kit, Sigma, France) based on the measurement of cellular protein content according to the manufacturer's instructions. Briefly, cells were fixed with 50% (wt/vol) TriChloroacetic Acid (TCA) at 4 °C (30 ul/well, final concentration 11.5%) for 1 h. After washing with dH2O cells were stained with 100 ul of 0.04% (wt/vol) SRB solution for 30 min and subsequently washed with 1% (vol/vol) acetic acid to remove unbound stain. Cellular protein bound dye was solubilized in 200 ul of 10 mM Tris base solution (pH 10.5) and the absorption was measured at 510 nm (Microplate reader, Tecan, Switzerland). The raw data was processed using GraphPad Prism 7.0 (GraphPad Software, San Diego, USA). Error bars in graphs represent SEM of two measurements.