For the in vivo efficacy studies, orthotopic TNBC tumors (MDA-MB-436 or MDA-MB-231) were established in 6- to 8-week-old female nude mice as described above. Tumors were grown to reach 100 mm3 for standard setting treatment or 500 mm3 for late-stage treatment. Then, tumor-bearing mice were randomly divided into various treatment groups (n ≥ 5 per group) and received treatment of PBS (sham), free Dox, IC1 Ab, IC1-MMAE, or IC1-MMAF at an equivalent dosage of 5 mg/kg per week for 3 weeks via tail vein injection. Tumor growth was monitored weekly using a caliper. At the end point, animals were euthanized with CO2 and orthotopic tumors were excised to measure their mass. In dosage-dependent experiments, three ascending IC1-MMAE dosages (1, 5, and 10 mg/kg) were evaluated in orthotopic TNBC tumors (MDA-MB-436) using the same protocol. Then, mice were euthanized with CO2 and 500 ul of mouse blood was collected immediately via cardiac puncture. Collected blood was incubated for 20 min at RT to allow clotting, and then mouse serum was isolated after centrifuging at 2000g for 10 min in a refrigerated centrifuge. Serum levels of ALT, AST, creatinine, and BUN were determined using their activity assay kits purchased from Sigma-Aldrich (St. Louis, MO, USA) with provided protocols. In MTD studies, the in vivo tolerability of IC1-MMAE was examined in 6- to 8-week-old female BALB/c mice. Healthy mice were randomized into various treatment groups (n = 10 per group) and received intravenous injections of IC1-MMAE at three ascending dosages (25, 50, and 75 mg/kg) for one injection. After injection, mouse body weight loss was used as the acute toxicity indicator and was closely monitored for up to 14 days.

For the in vivo efficacy studies, orthotopic TNBC tumors (MDA-MB-436 or MDA-MB-231) were established in 6- to 8-week-old female nude mice as described above. Tumors were grown to reach 100 mm3 for standard setting treatment or 500 mm3 for late-stage treatment. Then, tumor-bearing mice were randomly divided into various treatment groups (n ≥ 5 per group) and received treatment of PBS (sham), free Dox, IC1 Ab, IC1-MMAE, or IC1-MMAF at an equivalent dosage of 5 mg/kg per week for 3 weeks via tail vein injection. Tumor growth was monitored weekly using a caliper. At the end point, animals were euthanized with CO2 and orthotopic tumors were excised to measure their mass. In dosage-dependent experiments, three ascending IC1-MMAE dosages (1, 5, and 10 mg/kg) were evaluated in orthotopic TNBC tumors (MDA-MB-436) using the same protocol. Then, mice were euthanized with CO2 and 500 ul of mouse blood was collected immediately via cardiac puncture. Collected blood was incubated for 20 min at RT to allow clotting, and then mouse serum was isolated after centrifuging at 2000g for 10 min in a refrigerated centrifuge. Serum levels of ALT, AST, creatinine, and BUN were determined using their activity assay kits purchased from Sigma-Aldrich (St. Louis, MO, USA) with provided protocols. In MTD studies, the in vivo tolerability of IC1-MMAE was examined in 6- to 8-week-old female BALB/c mice. Healthy mice were randomized into various treatment groups (n = 10 per group) and received intravenous injections of IC1-MMAE at three ascending dosages (25, 50, and 75 mg/kg) for one injection. After injection, mouse body weight loss was used as the acute toxicity indicator and was closely monitored for up to 14 days.

Cells were seeded in a 96-well plate at a density of 5 × 103 cells per well and allowed to attach overnight. Then, cells were treated with four different ICAM1 ADCs (IC1-DM1, IC1-DM4, IC1-MMAE, and IC1-MMAF) at different drug concentrations ranging from 0 to 10 ug/ml using a serial dilution factor of 10. After cells were cultured for another 48 hours, the cytotoxicity was determined by CCK-8 assay. The plate was read at the absorbance wavelength of 450 nm using a Molecular Devices SpectraMax microplate reader (San Jose, CA, USA). Cell viability was determined by comparing the absorbance of cells incubated with drugs to that of the control cells incubated without the presence of the drug.

Cells were seeded in a 96-well plate at a density of 5 × 103 cells per well and allowed to attach overnight. Then, cells were treated with four different ICAM1 ADCs (IC1-DM1, IC1-DM4, IC1-MMAE, and IC1-MMAF) at different drug concentrations ranging from 0 to 10 ug/ml using a serial dilution factor of 10. After cells were cultured for another 48 hours, the cytotoxicity was determined by CCK-8 assay. The plate was read at the absorbance wavelength of 450 nm using a Molecular Devices SpectraMax microplate reader (San Jose, CA, USA). Cell viability was determined by comparing the absorbance of cells incubated with drugs to that of the control cells incubated without the presence of the drug.

Cells were seeded in a 96-well plate at a density of 5 × 103 cells per well and allowed to attach overnight. Then, cells were treated with four different ICAM1 ADCs (IC1-DM1, IC1-DM4, IC1-MMAE, and IC1-MMAF) at different drug concentrations ranging from 0 to 10 ug/ml using a serial dilution factor of 10. After cells were cultured for another 48 hours, the cytotoxicity was determined by CCK-8 assay. The plate was read at the absorbance wavelength of 450 nm using a Molecular Devices SpectraMax microplate reader (San Jose, CA, USA). Cell viability was determined by comparing the absorbance of cells incubated with drugs to that of the control cells incubated without the presence of the drug.