Abstract 2891: In vitro comparison of Kadcyla and Enhertu in breast cancer with varying HER2 expression: proliferation, internalization, bystander effects and toxicity
Authors: Veronica Bergo, Philipp Metzger, Valerie Oberhardt, Jan Eric Ehlert, Holger Weber
Journal: Cancer Research (2025)

Abstract HER2-positive breast cancer is characterized by overexpression of the HER2 receptor, driving aggressive tumor growth. Antibody-drug conjugates (ADCs), like trastuzumab emtansine (Kadcyla, T-DM1) and trastuzumab deruxtecan (Enhertu, T-DXd), are effective in targeting HER2-positive tumors, though their efficacy across varying HER2 levels remains under study. This study compares Kadcyla and Enhertu regarding cell proliferation, ADC internalization, and bystander cytotoxicity in cancer cell lines with high (SK-BR-3/SKOV-3), moderate (JIMT-1), and low (MDA-MB-231/MDA-MB-435) HER2 expression. It also examines off-site ADC toxicity in primary non-tumor cells caused by off-target payload delivery. The xCELLigence system, which works via label-free impedance measurements on living cells, was employed to monitor real-time cell proliferation and viability. Both, Kadcyla and Enhertu, eliminate tumor cells, effectively at nanomolar concentrations, correlating with HER2 expression levels. Interestingly, Kadcyla's effect appears at least 24 hours earlier than Enhertu, whereas the unconjugated antibody Trastuzumab itself has little to no effect on tumor cell growth. Low HER2-expressing cells were killed at 1 µM, the highest dose tested. ADC internalization was analyzed using pH-sensitive, fluorescently labeled Trastuzumab in flow cytometry and microscopy, assessing uptake by mean fluorescence intensity (MFI) in viable cells. This analysis allowed the comparison of the rate and extent of Trastuzumab uptake across the tested cell lines. A co-culture system evaluated bystander effects, with high HER2-expressing SKOV-3/SK-BR-3 cells co-cultured with luciferase-labeled HER2-negative LN-229 cells. After treatment with Kadcyla or Enhertu, luciferase assays were conducted to assess whether the cytotoxic payload released by the ADCs in HER2-positive cells could affect the viability of neighboring HER2-negative cells. Enhertu demonstrated a bystander effect, whereas Kadcyla, on the other hand, showed a more limited activity, reflecting the non-cleavable linker and the membrane-impermeable nature of its payload DM1. Toxicity analyses of Kadcyla and Enhertu on non-tumor cells, and especially on bone marrow, complement on-target investigations by revealing critical results about their safety profiles. While these ADCs are designed to selectively target tumor cells, off-site toxicity can affect healthy tissues and impair immune system function. Studies on hematopoietic stem cells are essential to assess potential adverse effects on hematopoietic regeneration and differentiation, aiming to reduce unintended immunosuppression. These findings support the optimization of ADC design, to enhance both selectivity and therapeutic windows. Citation Format: Veronica Bergo, Philipp Metzger, Valerie Oberhardt, Jan Eric Ehlert, Holger Weber. In vitro comparison of Kadcyla and Enhertu in breast cancer with varying HER2 expression: proliferation, internalization, bystander effects and toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2891.