Cytotoxicity assays performed in the presence of n501-MMAE and n501-alphaHSA-MMAE and their antibody control was assessed using cells plated at 5000 cells per well in medium with in flat bottom 96-well cell culture plates (NEST Biotechnology, China). After 12 h, tumor cells were added with serial dilutions of n501-alphaHSA-MMAE or n501-MMAE in triplicate wells and incubated for 72 h at 37°C in a humidified 5% CO2 atmosphere. After mixing CCK8 reagent and complete medium at a ratio of 1:9, the cell culture medium was removed and CCK8 working solution was added for another 1-3 h. The absorbance was measured by a microplate reader at 450 nm, and the cell viability ratio (%) was calculated using the following formula: (A sample - A blank)/ (A control - A blank) × 100%.

Cytotoxicity assays performed in the presence of n501-MMAE and n501-alphaHSA-MMAE and their antibody control was assessed using cells plated at 5000 cells per well in medium with in flat bottom 96-well cell culture plates (NEST Biotechnology, China). After 12 h, tumor cells were added with serial dilutions of n501-alphaHSA-MMAE or n501-MMAE in triplicate wells and incubated for 72 h at 37°C in a humidified 5% CO2 atmosphere. After mixing CCK8 reagent and complete medium at a ratio of 1:9, the cell culture medium was removed and CCK8 working solution was added for another 1-3 h. The absorbance was measured by a microplate reader at 450 nm, and the cell viability ratio (%) was calculated using the following formula: (A sample - A blank)/ (A control - A blank) × 100%.

Cytotoxicity assays performed in the presence of n501-MMAE and n501-alphaHSA-MMAE and their antibody control was assessed using cells plated at 5000 cells per well in medium with in flat bottom 96-well cell culture plates (NEST Biotechnology, China). After 12 h, tumor cells were added with serial dilutions of n501-alphaHSA-MMAE or n501-MMAE in triplicate wells and incubated for 72 h at 37°C in a humidified 5% CO2 atmosphere. After mixing CCK8 reagent and complete medium at a ratio of 1:9, the cell culture medium was removed and CCK8 working solution was added for another 1-3 h. The absorbance was measured by a microplate reader at 450 nm, and the cell viability ratio (%) was calculated using the following formula: (A sample - A blank)/ (A control - A blank) × 100%.

Cytotoxicity assays performed in the presence of n501-MMAE and n501-alphaHSA-MMAE and their antibody control was assessed using cells plated at 5000 cells per well in medium with in flat bottom 96-well cell culture plates (NEST Biotechnology, China). After 12 h, tumor cells were added with serial dilutions of n501-alphaHSA-MMAE or n501-MMAE in triplicate wells and incubated for 72 h at 37°C in a humidified 5% CO2 atmosphere. After mixing CCK8 reagent and complete medium at a ratio of 1:9, the cell culture medium was removed and CCK8 working solution was added for another 1-3 h. The absorbance was measured by a microplate reader at 450 nm, and the cell viability ratio (%) was calculated using the following formula: (A sample - A blank)/ (A control - A blank) × 100%.

Cytotoxicity assays performed in the presence of n501-MMAE and n501-alphaHSA-MMAE and their antibody control was assessed using cells plated at 5000 cells per well in medium with in flat bottom 96-well cell culture plates (NEST Biotechnology, China). After 12 h, tumor cells were added with serial dilutions of n501-alphaHSA-MMAE or n501-MMAE in triplicate wells and incubated for 72 h at 37°C in a humidified 5% CO2 atmosphere. After mixing CCK8 reagent and complete medium at a ratio of 1:9, the cell culture medium was removed and CCK8 working solution was added for another 1-3 h. The absorbance was measured by a microplate reader at 450 nm, and the cell viability ratio (%) was calculated using the following formula: (A sample - A blank)/ (A control - A blank) × 100%.

Cytotoxicity assays performed in the presence of n501-MMAE and n501-alphaHSA-MMAE and their antibody control was assessed using cells plated at 5000 cells per well in medium with in flat bottom 96-well cell culture plates (NEST Biotechnology, China). After 12 h, tumor cells were added with serial dilutions of n501-alphaHSA-MMAE or n501-MMAE in triplicate wells and incubated for 72 h at 37°C in a humidified 5% CO2 atmosphere. After mixing CCK8 reagent and complete medium at a ratio of 1:9, the cell culture medium was removed and CCK8 working solution was added for another 1-3 h. The absorbance was measured by a microplate reader at 450 nm, and the cell viability ratio (%) was calculated using the following formula: (A sample - A blank)/ (A control - A blank) × 100%.

Cytotoxicity assays performed in the presence of n501-MMAE and n501-alphaHSA-MMAE and their antibody control was assessed using cells plated at 5000 cells per well in medium with in flat bottom 96-well cell culture plates (NEST Biotechnology, China). After 12 h, tumor cells were added with serial dilutions of n501-alphaHSA-MMAE or n501-MMAE in triplicate wells and incubated for 72 h at 37°C in a humidified 5% CO2 atmosphere. After mixing CCK8 reagent and complete medium at a ratio of 1:9, the cell culture medium was removed and CCK8 working solution was added for another 1-3 h. The absorbance was measured by a microplate reader at 450 nm, and the cell viability ratio (%) was calculated using the following formula: (A sample - A blank)/ (A control - A blank) × 100%.