Fig. 2: HER2-independent DXd payload release by Cathepsin L (CTSL) and its expression in human breast cancer biopsies.

a MDA-MB-231 tumors treated with T-DXd (4 mg/kg) were co-injected with fivefold higher dose of trastuzumab (20 mg/kg) to assess T-DXd efficacy after HER2 binding competition. Mixed-effects analysis (REML) with Tukey’s multiple comparisons (n = 10). Arrows indicate treatment administered. b Liquid chromatography-mass spectrometry (LC-MS) quantification of DXd molecules in tumors treated with T-DXd (10 mg/kg) for 3 days. DXd levels per tumor mass were quantified. One-way ANOVA with Tukey’s multiple comparisons test (n = 13). c Systemic DXd payload levels in plasma from same treated animals in (b). DXd levels (ng) per plasma volume (mL) were quantified. No statistical significance between plasma groups was observed (n = 13). d T-DXd was incubated with recombinant human CTSL or CTSB for 6 h in low pH assay buffer as described in Methods. Released DXd levels were quantified by LC-MS. e Cytotoxicity of cleaved T-DXd was assessed on MDA-MB-468 cells. f Immunohistochemistry analysis of primary core needle biopsies from BC patients. Representative HER2 expression (top row) is shown. CTSL expression and localization in the same patient’s biopsy are shown (bottom row). Positive staining in tumor beds (red arrows) and stromal compartments (black arrows) are indicated. g–i Tissue microarray comprising 321 invasive BC, 20 DCIS, and 49 normal breast samples were assessed for CTSL expression via IHC. Pixelwise H-scores were used for quantification by QuPath. One-way ANOVA with Tukey’s multiple comparisons test. g CTSL expression differences in invasive BC, DCIS, and normal breast. h CTSL expression among lymph node-negative, lymph node-positive, or metastatic BC. i CTSL expression among BC with different HER2 IHC scores.