Fig 2: Molecular design and conjugation strategy for generating dual-drug ADCs.MTGase-mediated conjugation of bi-functional branched linkers and following orthogonal click reactions with two payload modules afford homogeneous dual-drug ADCs with defined DARs (magenta circle: MMAE; yellow triangle: MMAF). Preparation of anti-HER2 ADC with a DAR of MMAE/F 4 + 2 is shown as an example. The glutamic acid–valine–citrulline (GluValCit)–PABC linker ensures in vivo stability, while allowing for quick and traceless release of payloads upon internalization and following cathepsin-mediated cleavage in lysosomes. DAR, drug-to-antibody ratio; DBCO, dibenzocyclooctyne; MTGase, microbial transglutaminase; PABC, p-aminobenzyloxycarbonyl; PEG, polyethylene glycol; TCO, trans-cyclooctene.

Fig 3: Construction and characterization of dual-drug ADCs.a Deconvoluted ESI-mass spectra. First panel: intact N297A anti-HER2 mAb (trastuzumab mutant). Second panel: antibody–branched linker conjugate. Third panel: intermediate after conjugation with TCO–MMAF modules. Fourth panel: highly homogeneous dual-drug ADC with a DAR of 4 + 2 (MMAE + MMAF). *Fragment ions detected in ESI-MS analysis. b Diagrams illustrating dual-drug and single-drug ADCs prepared and evaluated in this study. Magenta circle: MMAE; yellow triangle: MMAF. See Supplementary Notes for characterization details. c Hydrophobic interaction chromatography (HIC) analysis of ADCs under physiological conditions (phosphate buffer, pH 7.4). The number of conjugated MMAE has a greater contribution to ADC hydrophobicity than does MMAF. d Overlay traces of size-exclusion chromatography (SEC) after incubating each conjugate in PBS (pH 7.4) at 37 °C for 28 days. MMAE/F 2 + 4 ADC (light purple), MMAE/F 2 + 2 ADC (dark purple), MMAE DAR 4 ADC (green), MMAF DAR 4 ADC (magenta), and MMAE/F 4 + 2 ADC (cyan). Retention times of all major peaks were similar (12 min). No significant aggregation was detected in either case.

Fig 4: Evaluation of payload delivery efficiency by the dual conjugate format.a Structures of anti-HER2 AF488/Cy5 dual-dye conjugate (DOL of 2 + 2) and single-dye variants (DOL of 2). b, c Whole tumor fluorescence imaging of JIMT-1/MDA-MB-231 (4 : 1) admixed tumors 24 h after intravenous administration of mAb–AF488/Cy5 (3 mg kg−1, black circle) or a 1 : 1 cocktail of mAb–AF488 and mAb–Cy5 (3 mg kg−1 each, black square) into tumor-bearing female NU/J mice (n = 3 per group). Fluorescence images of the whole tumors (b) and semi-quantification (c) detected using a 700 nm channel. d–f Fluorescence microscopic imaging of the tumor tissues. Fluorescence images of the frozen sections (AF488 and Cy5 channel, scale bar: 300 µm (d) and semi-quantification (AF488 channel (e) and Cy5 channel (f)). For tissue section analysis, six areas were randomly selected from the tissue sections and normalized signal intensity (intensity in each region of interest divided by area) was calculated using ImageJ software. Data are presented as mean values ± SEM (n = 6). For statistical analysis, a two-tailed Welch’s t-test was used. This experiment was repeated twice independently with similar results. Source data are available as a Source Data file. AF488, Alexa Fluor® 488; DOL, degree of labeling.

Fig 5: In vivo antitumor activity in the low-HER2 heterogeneous HCC1954-TDR model.a Antitumor activity of each ADC in the HCC1954-TDR orthotopic breast tumor model (female NU/J mice, n = 11 for vehicle; n = 12 for other groups). Once tumors reached an average volume of 125 mm3 (indicated as day 0), mice were administered with a single dose of a 1 : 1 mixture of MMAF DAR 4 and MMAE DAR 4 ADCs (magenta open triangle, 1 mg kg-1 each), MMAE/F 4 + 2 ADC (cyan square, 1 mg kg-1), or vehicle control (black circle). All animals were killed 24 days post ADC injection. Data are presented as mean values ± SEM. b Tumors collected 24 days post injection of each ADC. Scale bar: 1 cm. c Weight of the collected tumors at Day 24. Lines represent mean values. For statistical analysis, a two-tailed Welch’s t-test was used. Source data are available as a Source Data file.