Abstract 6746: Multi-modal assessment of an anti-HER2 (trastuzumab) antibody-drug conjugate screening assay for breast cancer models

Abstract Antibody-drug conjugates (ADCs) represent groundbreaking advancement in the cancer treatment landscape. ADCs combine the specific binding capacity of a monoclonal antibody with a cytotoxic payload that can kill target and bystander cells. The antibody moiety of ADCs confers payloads with the ability to target cancer cells with minimal damage to healthy tissues. A key consideration in defining appropriate breast cancer treatment regimen is the expression level of human epidermal growth factor receptor 2 (HER-2), targeted with the antibody Trastuzumab. Breast cancers are classified as high, low, or negative for HER-2, which dictates the therapeutic approach. Trastuzumab alone may not be an effective treatment because tumors can either be initially resistant or develop resistance over time. Around 70% of patients experience resistance within the first year of treatment. To overcome this, ADCs such as Trastuzumab-Emtansine (DM1) or Trastuzumab-Deruxtecan (Dxd) can be used at lower doses, reducing the risk of resistance and potentially enhancing treatment efficacy. Assessment of Trastuzumab ADCs in multiple murine breast cancer models can be costly, but in vitro evaluation of various cancer histotypes facilitates the selective identification of the best murine model. Having a robust screening tool is an important asset. To assess Trastuzumab ADCs, we screened five breast cancer cells (BT-474, Hs 578T, MDA-MB-231, MDA-MB-361, SK-BR-3) with varying levels of HER-2 expression. They were evaluated for binding efficiency, competitive inhibition, and cytotoxicity of ADCs. Additionally, the ability of the ADC to kill bystander cells (HER-2 low or negative cells) was assessed using two bystander assay methods. The half maximal effective concentration (EC50) values for Trastuzumab and Trastuzumab-Dxd were measured on two cell lines. The HER-2 high cell line had a tenfold higher EC50 value. Competitive inhibition of Trastuzumab-ADC binding was tested with a PE labeled Trastuzumab on two cell lines. The Trastuzumab-ADC was able to outcompete Trastuzumab on HER-2 high cells but not on HER-2 low/negative cells. Proliferation assays on multiple cell lines were used to assess the ability of Trastuzumab-DM1 to inhibit cell growth in comparison to DM1 (payload alone) or Trastuzumab (antibody alone). Overall, Trastuzumab-DM1 was better able to inhibit growth of HER-2 positive cells compared to payload or antibody alone. Two ADC bystander assays were developed and tested. The first involved flow cytometry and the second a luciferase assay. In both assays, bystander cells were more effectively targeted only in the presence of HER-2 positive cells. The ability of ADCs to target bystander cells suggest that even in heterogenous HER-2 expressing tumors, these therapies may still be effective. Our cutting-edge approaches provide precise insights into the activity and efficacy of Trastuzumab ADCs. Citation Format: Michael Steffey, Stacey Roys, Anita J. Zaitouna, Philip Lapinski, Kerry-Ann Bright, Sheri Barnes, David Draper, Scott Wise. Multi-modal assessment of an anti-HER2 (trastuzumab) antibody-drug conjugate screening assay for breast cancer models [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 6746.