Having ADCs with cleavable and non-cleavable linkers in place, we next assessed there in vitro cytotoxicity activities using SSEA4-expressing ovarian SKVO3 cells and breast cancer cells SKBR3 with a negligible expression of SSEA4 and 293T cells as a control. SK-OV3, SKBR3, and 293T cells (2×103 cells/well) were incubated with ADC-1, ADC-2, and chMC-813-70 for 72 h and then treated with MTS at 37 °C. The conversion of the tetrazolium salt MTS (3- (4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium) to a purple formazan, as a measure of the percentage of viable cells, was measured by absorbance at 490 nm after 4 h. The untreated cells' absorbance was considered 100 % survival (Figure 4).

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cytotoxic effects of aTCRA6 DAR2 and aTCRA6 DAR52-8 ADCs were estimated by exposing Jurkat WT and Jurkat TCRA6 T cells to different ADC concentrations. Cell viability was analyzed 72 h post ADC addition by a fluorometric method using CellTiter-Blue Cell Viability Assay (Promega, Wisconsin, USA) according to the manufacturer's instructions. Briefly, cells were seeded in a 96-well plate (1 × 104 cells/well) with the desired ADC concentrations ranging from 0.06-400 nM (for DAR2 ADC) or 0.005-30 nM (for DAR52-8 ADC) in a serial dilution. After 72 h, redox dye (resazurin) was added to the cells, and the plate was incubated for 3-4 h. Fluorescence of resorufin (560Ex/590Em) was recorded using CLARIOstar plus microplate reader (BMG LABTECH, Offenburg, Germany). Cell proliferation was normalized to untreated control cell fluorescence values. The resulting curves were fitted with a variable slope four-parameter fit, and EC50s were calculated using GraphPad Prism.

Cytotoxic effects of aTCRA6 DAR2 and aTCRA6 DAR52-8 ADCs were estimated by exposing Jurkat WT and Jurkat TCRA6 T cells to different ADC concentrations. Cell viability was analyzed 72 h post ADC addition by a fluorometric method using CellTiter-Blue Cell Viability Assay (Promega, Wisconsin, USA) according to the manufacturer's instructions. Briefly, cells were seeded in a 96-well plate (1 × 104 cells/well) with the desired ADC concentrations ranging from 0.06-400 nM (for DAR2 ADC) or 0.005-30 nM (for DAR52-8 ADC) in a serial dilution. After 72 h, redox dye (resazurin) was added to the cells, and the plate was incubated for 3-4 h. Fluorescence of resorufin (560Ex/590Em) was recorded using CLARIOstar plus microplate reader (BMG LABTECH, Offenburg, Germany). Cell proliferation was normalized to untreated control cell fluorescence values. The resulting curves were fitted with a variable slope four-parameter fit, and EC50s were calculated using GraphPad Prism.

Cytotoxic effects of aIgM ADCs were evaluated by exposing IgM+ lymphoma B cells or off-target (IgM-) cells to different ADC concentrations. Cell viability was analyzed 72 h post ADC addition by a colorimetric method using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Briefly, cells were seeded (1x104 cells/well) in a 96-well plate with the desired antibody concentrations ranging from 0.014-90 nM in a serial dilution. After 72 h, MTS solution was added to the cells and plate was incubated for 2 h. Absorption was measured at 490 nm using CLARIOstar plus microplate reader (BMG LABTECH). Cell proliferation was normalized to untreated control cell absorption values. The resulting curves were fitted with a variable slope four-parameter fit and EC50s were calculated using GraphPad Prism.

Cytotoxic effects of aTCRA6 DAR2 and aTCRA6 DAR52-8 ADCs were estimated by exposing Jurkat WT and Jurkat TCRA6 T cells to different ADC concentrations. Cell viability was analyzed 72 h post ADC addition by a fluorometric method using CellTiter-Blue Cell Viability Assay (Promega, Wisconsin, USA) according to the manufacturer's instructions. Briefly, cells were seeded in a 96-well plate (1 × 104 cells/well) with the desired ADC concentrations ranging from 0.06-400 nM (for DAR2 ADC) or 0.005-30 nM (for DAR52-8 ADC) in a serial dilution. After 72 h, redox dye (resazurin) was added to the cells, and the plate was incubated for 3-4 h. Fluorescence of resorufin (560Ex/590Em) was recorded using CLARIOstar plus microplate reader (BMG LABTECH, Offenburg, Germany). Cell proliferation was normalized to untreated control cell fluorescence values. The resulting curves were fitted with a variable slope four-parameter fit, and EC50s were calculated using GraphPad Prism.

Cytotoxic effects of aTCRA6 DAR2 and aTCRA6 DAR52-8 ADCs were estimated by exposing Jurkat WT and Jurkat TCRA6 T cells to different ADC concentrations. Cell viability was analyzed 72 h post ADC addition by a fluorometric method using CellTiter-Blue Cell Viability Assay (Promega, Wisconsin, USA) according to the manufacturer's instructions. Briefly, cells were seeded in a 96-well plate (1 × 104 cells/well) with the desired ADC concentrations ranging from 0.06-400 nM (for DAR2 ADC) or 0.005-30 nM (for DAR52-8 ADC) in a serial dilution. After 72 h, redox dye (resazurin) was added to the cells, and the plate was incubated for 3-4 h. Fluorescence of resorufin (560Ex/590Em) was recorded using CLARIOstar plus microplate reader (BMG LABTECH, Offenburg, Germany). Cell proliferation was normalized to untreated control cell fluorescence values. The resulting curves were fitted with a variable slope four-parameter fit, and EC50s were calculated using GraphPad Prism.