Abstract P1-11-10: Reversible versus irreversible tyrosine kinase inhibitors (TKIs) combined with antibody-drug conjugates (ADCs) in HER2-positive (HER2+) breast cancer (BC) cell lines
Authors: Ashfield Niall , Mahdi Amira F. , Conlon Neil T. , Crown John , Collins Denis M. 
Published: March 2023

Abstract
Background: Targeting the human epidermal growth factor receptor (HER) family of tyrosine kinase receptors with small molecule TKIs lapatinib, neratinib and tucatinib, monoclonal antibodies like trastuzumab and more recently with ADCs like T-DM1 and T-DXd has resulted in improved survival rates for patients with HER2+ BC. TKIs are primarily utilised in trastuzumab-refractory disease. Neratinib, and the pan-HER TKI afatinib, bind irreversibly to all members of the HER family, while lapatinib binds reversibly to HER2 and EGFR, and tucatinib binds reversibly to HER2 alone. Previous work in our lab has shown that TKIs like lapatinib are capable of modulating tumour surface HER2 levels and increasing trastuzumab load on tumours. Thus, there is a rationale for the use of TKI/ADC combinations in HER2+ BC. Using innately trastuzumab-resistant HER2+ BC cell lines, this in vitro study aims to assess the anti-proliferative potential of HER2-targeting ADCs T-DM1 or T-DXd in combination with the TKIs afatinib, lapatinib, neratinib or tucatinib.
Methods: HCC1569 and HCC1954 cells (HER2+, estrogen receptor (ER)-negative; innately trastuzumab-resistant) were grown in RPMI1640/10% FBS at 37°C and 5% CO2. The anti-proliferative effects of afatinib, lapatinib, neratinib and tucatinib; of T-DM1 and T-DXd; and of each TKI/ADC combination thereof were assessed in these cells via 5-day acid phosphatase. ADCs were obtained from Saint Vincent’s University Hospital, Dublin, and TKIs were purchased from commercial sources. Calcusyn software was used to generate IC50 values and combination index (CI) values at ED50. CI values &gt; 1 represent an antagonistic combination, CI values = 1 are additive, and CI values &lt; 1 are synergistic. All assays were carried out in triplicate.
Results: The HCC1569 cell line was more sensitive to all four TKIs (IC50 values were 12.8 ± 0.3, 453.8 ± 47.1 nM, 4.7 ± 1.4 and 381.5 ± 37.3 nM for afatinib, lapatinib, neratinib and tucatinib respectively) compared to the HCC1954 cell line (IC50 22.3 ± 2.8, 652.4 ± 36.0, 57.0 ± 6.3 and 2365.1 ± 185.3 nM for afatinib, lapatinib, neratinib and tucatinib respectively). Despite their innate trastuzumab resistance, both cell lines displayed high sensitivity to T-DM1 (IC50 25.2 ± 8.7 ng/mL for HCC1569 and IC50 30.4 ± 3.2 ng/mL for HCC1954) and to T-DXd (IC50 16.0 ± 3.2 ng/mL for HCC1569 and IC50 36.8 ± 8.7 ng/mL for HCC1954). In both cell lines, co-treatment with ADCs and the irreversible TKIs afatinib and neratinib resulted in additive or synergistic responses, while the combination of the ADCs with the reversible TKIs lapatinib and tucatinib resulted in mild to moderate antagonism (Table 1).
Conclusions: In this pre-clinical study, T-DM1 and T-DXd consistently exhibited antagonistic interactions with reversible HER2-targeting TKIs, and synergy/additivity in combination with the irreversible TKIs tested. Future work will explore the underlying mechanisms of this observed synergy and antagonism, including impacts of TKIs on HER2 expression and real-time ADC internalisation rates.
Table 1. CI values +/- Std Dev for each TKI/ADC treatment combination in HCC1569 and HCC1954 cells
Citation Format: Niall Ashfield, Amira F. Mahdi, Neil T. Conlon, John Crown, Denis M. Collins. Reversible versus irreversible tyrosine kinase inhibitors (TKIs) combined with antibody-drug conjugates (ADCs) in HER2-positive (HER2+) breast cancer (BC) cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-10.