A xenograft model derived from cell lines was established by unilateral subcutaneous injection of 1×10^6 HT-29 cells into female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 2 mg/kg. The treatment was given twice a week for 4 weeks. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from cell lines was established by unilateral subcutaneous injection of 1×10^6 HT-29 cells into female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 0.4 mg/kg. The treatment was given twice a week for 4 weeks. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from cell lines was established by injecting 1×10^7 Jeko cells subcutaneously into the left side of female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE at doses of 2.5 mg/kg and 3.5 mg/kg was administered on the 15th and 29th days respectively. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from cell lines was established by unilateral subcutaneous injection of 1×10^6 A549 cells into female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 10 mg/kg. The treatment was given twice a week for 3 weeks. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from cell lines was established by injecting 1×10^7 Jeko cells subcutaneously into the left side of female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 2.5 mg/kg. The treatment was given once a week for 3 weeks. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from the cell line was established by injecting 1×10^6 MDA-MB-231 cells subcutaneously into the left side of female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 5 mg/kg. The treatment was given once a week for 3 weeks. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from cell lines was established by unilateral subcutaneous injection of 1×10^6 HT-29 cells into female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 10 mg/kg. The treatment was given twice a week for 4 weeks. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from the cell line was established by injecting 1×10^6 NCI-N87 cells subcutaneously into the left side of female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 5 mg/kg. The treatment was given once a week for 3 weeks. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from cell lines was established by injecting 1×10^7 Jeko cells subcutaneously into the left side of female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 10 mg/kg. The treatment was given once a week for 3 weeks. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from the cell line was established by injecting 1×10^6 MDA-MB-231 cells subcutaneously into the left side of female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 10 mg/kg. The treatment was given once a week for 3 weeks. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from the cell line was established by injecting 1×10^7 MDA-MB-468 cells subcutaneously into the left side of female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 5 mg/kg. The treatment was given once a week for 4 weeks. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from the cell line was established by injecting 1×10^6 NCI-N87 cells subcutaneously into the left side of female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 10 mg/kg. The treatment was given once a week for 3 weeks. The tumor inhibition rate was calculated at the end of the experiment.

A xenograft model derived from the cell line was established by injecting 1×10^7 MDA-MB-468 cells subcutaneously into the left side of female Balb/C nude mice. When the tumor volume reached approximately 100 mm^3, B31-H3L3-MMAE was administered at a dose of 10 mg/kg. The treatment was given once a week for 4 weeks. The tumor inhibition rate was calculated at the end of the experiment.

NCI-H1944 cells were added with ADC samples, and the drug concentration gradient was diluted to 500, 158, 50, 15.8, 5, 1.58, 0.5, 0.158, and 0.05 nM. The cells were cultured at 37°C with 5% CO2 for 72 hours. Then, 10 uL of LCK8 (Bimake, B34304) was added to each well, and the cells were incubated at 37°C for 1 hour. The data were read at OD450 using a microplate reader.

MDA-MB-468 cells were added with ADC samples, and the drug concentration gradient was diluted to 500, 158, 50, 15.8, 5, 1.58, 0.5, 0.158, and 0.05 nM. The cells were cultured at 37°C with 5% CO2 for 72 hours. Then, 10 uL of LCK8 (Bimake, B34304) was added to each well, and the cells were incubated at 37°C for 1 hour. The data were read at OD450 using a microplate reader.

Jeko-1 cells were co-cultured with ADC samples, and the drug concentration gradient was diluted to 500, 158, 50, 15.8, 5, 1.58, 0.5, 0.158, and 0.05 nM. The cells were cultured at 37°C with 5% CO2 for 72 hours. Then, 10 uL of LCK8 (Bimake, B34304) was added to each well, and the reaction was incubated at 37°C for 1 hour. The data were read at OD450 using a microplate reader.