Animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Seoul National University. A heterogeneous tumor model was established by subcutaneously injecting a mixture of 1 × 107 JIMT-1 cells and 2.5 × 106 MDA-MB-231 cells in 100 uL PBS into the dorsal flank of 6-week-old female NU/J mice (OrientBio) [29]. This 4:1 co-implantation reflects the diverse composition often observed in advanced HER2-positive breast cancer while ensuring enough HER2-negative cells to model intratumor heterogeneity [29]. When tumors reached approximately 150 mm3, mice were randomly assigned to different experimental groups and received intravenous injections of PBS (Control), IgG DEVD ADC, HER2 DEVD ADC, or T-Dxd (trastuzumab deruxtecan) at a dose of 5 mg/kg weekly for four weeks. This 5 mg/kg dose aligns with the range commonly used in prior preclinical trastuzumab-based ADC evaluations [7,12] and follows recent analysis [30] suggesting that efficacy in mice is most meaningful when the mg/kg dose approximates the maximum tolerated dose seen in human trials. Tumor volumes and body weights were measured every three days. Tumor volume was calculated using the ellipsoidal formula: volume = (length × width2)/2. The tumor growth inhibition (%TGI) was calculated using the formula: %TGI = [1 - (Tt/T0)/ (Ct/C0)]/[1- (C0/Ct)] × 100, where Tt and Ct are the mean tumor volumes of the treatment and control groups at the endpoint, and T0 and C0 are the initial tumor volumes. Mice were euthanized when tumors exceeded 1000 mm3 or if severe distress was observed. Tumors were harvested for histological analysis.