Figure 3.
In vitro cytotoxicity of anti-CDCP1 ADCs: A. Comparative analysis of ch10D7-MMAE and 10D7-MMAE ADC binding affinity to CDCP1 ECD by SPR analysis. Top: SPR-derived sensograms of ch10D7-MMAE and 10D7-MMAE binding to various concentrations of recombinant CDCP1-ECD. Bottom: Table summarizing association (Ka), dissociation (Kd) and affinity (KD) constants of ch10D7-MMAE and 10D7-MMAE to CDCP1-ECD. B. Quantitative analysis of growth inhibition of cancer cells by ADCs. Cancer cells (4,000 cells/well) were treated for 6 h with the respective ADC (0 - 2000 ng/ml) then grown for a further 72 h in complete medium. Cell growth was quantified by absorbance measurements at 490 nm of wells incubated with the CellTiter AQueous One Solution Reagent. Data are presented as mean of relative cell growth (compared to untreated cells) +/- SD from three independent experiments. C. Qualitative analysis of the impact of ADCs on cancer cell colony formation. Cell lines were treated for 6 h with the respective ADC (500 - 1000 ng/ml) before plating at low density (500 cells/well) in complete medium and grown for a further 10 to 14 days when colonies were fixed using PFA and stained with crystal violet. Representative images of colonies are shown. D. Examination of correlation between cell surface CDCP1 and cell response to ADC ch10D7-MMAE. Left: The number of fluorescently labelled anti-CDCP1 antibodies bound per cell was evaluated by flow cytometry against a panel of 49 cancer cell lines using an anti-CDCP1 antibody conjugated with PE (CD318-PE). The number of fluorescently labelled anti-CDCP1 antibodies bound/cell was interpolated from a standard curve generated from known numbers of Quantibright beads. Results are expressed as median of antibodies/cell from at least 5,000 cells. Right: Correlation between the potency of ch10D7-MMAE ADC (represented by GI50 values for each cell line) versus the number of fluorescently labelled anti-CDCP1 antibodies bound/cell. MW, Molecular weight.