Abstract 1564: Receptor quantification in the context of human epidermal growth factor receptor 2 (HER2) antibody-drug conjugates
Authors: Mandy W. Drossaert-Smeets, Eef F. Smits, Milan M. Bolwerk, Janneke J. Melis, Dimitri Pappaioannou, Guido J. Zaman
Journal: Cancer Research (2025)

Abstract Antibody-drug conjugates (ADCs), comprised of cancer cell-specific antibodies linked to cytotoxic drugs, represent an innovative and promising class of anticancer agents. ADCs often rely on high receptor numbers for target binding, followed by internalization and release of the payload to exert a tumor-targeted, intracellular effect. Determination of the exact number of receptors per cell is therefore highly valuable in the context of ADCs. Here, we focused on the quantification of the human epidermal growth factor receptor 2 (HER2), since expression of this target is often amplified in various tumor types. Two HER2-targeting ADCs, i.e., ado-trastuzumab emtansine (T-DM1) and fam-trastuzumab deruxtecan-nxki (T-DXd), have already been approved for clinical use, while roughly twenty HER2-targeting ADCs are currently under investigation. The development and improvement of ADCs could benefit from in vitro studies investigating the internalization, cytotoxic effect and possible bystander effect of ADCs related to receptor numbers. The number of HER2 per cell was quantified in a panel of thirteen cancer cell lines originating from different cancer types. The cell lines BxPC-3, CAL-27, COLO 205, HCC1954, HEC-251, K-562, MOLM-13, NCI-H23, RT-112, SK-BR-3, SK-OV-3, SW837 and U-87MG ATCC were cultured for one week in the supplier’s recommended medium. Quantification of the number of surface-expressed receptors was performed with flow cytometry using Quantibrite PE beads and PE-conjugated ErbB2/HER2 antibody. The effect of T-DM1 and T-DXd on the viability of HER2 high-, intermediate- and low-expressing cells was determined in proliferation assays in 9-point dose ranges. Three independent flow cytometry experiments revealed that the breast and ovarian cancer cell lines HCC1954, SK-BR-3, and SK-OV-3 expressed the highest number of HER2 per cell, ranging between 20, 000 and 53, 000 receptors. Intermediate expression was observed on the COLO 205, HEC-251, RT-112 and SW837 cell lines, with a range of 1800 to 3800 receptors per cell. BxPC-3, CAL-27, K-562, MOLM-13, NCI-H23 and U-87MG ATCC belong to the group with the lowest or no expression of HER2, ranging up to 400 receptors per cell. T-DM1 and T-DXd induced efficient cell death in cells expressing high receptor numbers, while little or no effect was observed in cell lines with intermediate, low or no expression of HER2. Since high target expression is correlated with higher efficacy for several ADCs, as shown for T-DM1 and T-DXd is our study, this indicates that HCC1954, SK-BR-3 or SK-OV-3 cells could serve as an attractive model to study HER2 internalization. Moreover, co-cultures of HER2-positive and HER2-negative cells could aid in the investigation of ADC bystander effect. This research demonstrates the importance of receptor number quantification in the context of ADCs and may therefore contribute to future ADC development. Citation Format: Mandy W. Drossaert-Smeets, Eef F. Smits, Milan M. Bolwerk, Janneke J. Melis, Dimitri Pappaioannou, Guido J. Zaman. Receptor quantification in the context of human epidermal growth factor receptor 2 (HER2) antibody-drug conjugates [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 1564.