Fig. 5. In vivo antitumor activity of dual-drug ADCs in the JIMT-1/MDA-MB-231 admixed model.

a HER2 expression on tumors consisting of either HER2-positive JIMT-1 or HER2-negative MDA-MB-231 cells, or both (4 : 1 ratio at the time of implantation). Immunohistochemistry for HER2 was performed on frozen sections of each tumor. Scale bar: 200 µm. This experiment was repeated twice independently with similar results. b–e Antitumor activity (b, d) and survival benefit (c, e) of each ADC in the JIMT-1/MDA-MB-231 admixed orthotopic breast tumor model (female NU/J mice, n = 4 for vehicle; n = 6 for MMAE DAR 4 at 1 mg kg⁻¹; n = 5 for all other groups). Tumor-bearing mice were treated with each ADC at 3 mg kg⁻¹ (b, c) or 1 mg kg⁻¹ (d, e). At day 8 post transplantation (indicated with a black arrow), mice were administered with a single dose of MMAF DAR 4 ADC (magenta square), MMAE DAR 4 ADC (green triangle), MMAE DAR 6 ADC (black open square), a 1 : 1 mixture of MMAF DAR 4 and MMAE DAR 4 ADCs (magenta open triangle, 3 or 1 mg kg⁻¹ each), MMAE/F 2 + 4 ADC (light purple diamond), MMAE/F 4 + 2 ADC (cyan square), or vehicle control (black circle). All animals other than the ones that were found dead or achieved complete remission were killed at the pre-defined humane endpoint (see “Methods”), which were counted as deaths. Data are presented as mean values ± SEM. For statistical analysis, a two-tailed Welch’s t-test (for tumor volume) and a log-rank test (for survival curve) were used. To control the family-wise error rate in multiple comparisons, crude P-values were adjusted by the Holm–Bonferroni method (see Supplementary Table 4 for details). f HER2 expression of regrown tumors after treatment with MMAE DAR 4 ADC (1 mg kg⁻¹), a 1 : 1 mixture of MMAF DAR 4 and MMAE DAR 4 ADCs (1 mg kg⁻¹ each), and MMAE/F 4 + 2 ADC (1 mg kg⁻¹). Each tumor was collected when its size reached 1000 mm³ and fixed with 4% PFA. Immunohistochemistry for HER2 was performed on frozen sections. Scale bar: 100 µm. This experiment was repeated twice independently with similar results. Source data are available as a Source Data file.