Abstract Introduction: Human epidermal growth factor 2 (HER2/ErbB2) is overexpressed in approximately 20% of breast cancers. The HER2-targeted tyrosine kinase inhibitors (TKIs) neratinib, lapatinib and tucatinib are all FDA-approved for the treatment of HER2-positive breast cancer. All three inhibit the HER2 kinase domain but differ in binding characteristics (the irreversible neratinib vs reversible lapatinib and tucatinib) and their affinity for other HER family members. The aim of this study was to carry out kinetic analysis of the effect of the three TKIs on HER family signalling, cellular proliferation, and apoptosis induction in a cell model of breast cancer. Methods: In this study, the anti-proliferative effects of the three TKIs were assessed in the HER2-amplified breast cancer cell line SKBR3 by acid phosphatase assay (5-day endpoint assay) and with the IncuCyte→ live-cell imaging system over 120 hours. TKI IC50 values were calculated using CalcuSyn software. Apoptotic cells were detected every 3 hours for 5 days using the Incucyte→ Caspase 3/7 dye. Western blotting was used to investigate the time course effects (15 min, 6 h, 24 h, 72 h) of the TKIs on the expression and phosphorylation of EGFR, HER2 and on their downstream effectors ERK1/2 and Akt. Results: All three TKIs displayed nanomolar activity against SKBR3 cells. Neratinib showed the greatest anti-proliferative effect (IC50 = 3.4 ± 1.1 nM), followed by tucatinib (IC50 = 37.5 ± 18.4 nM) and lapatinib (IC50 = 51.0 ± 23.0 nM). 50 nM neratinib induced higher activation of caspase 3/7 than 500 nM lapatinib or 500 nM tucatinib. Total levels of HER2 and EGFR were increased in the presence of lapatinib and tucatinib, but decreased in the presence of neratinib. The three drugs (500 nM) significantly inhibited the activation of EGFR and HER2 at 15 min. From 6 h to 72 h, treatment with 5 nM and 50 nM neratinib resulted in greater inhibition of phosphorylated HER2 and EGFR, compared to the equivalent concentration of lapatinib or tucatinib. All three TKIs (500 nM) inhibited the activity of phospho-Akt at the earliest timepoint (15 min), but a strong impact on downstream ERK1/2 phosphorylation was not observed until the 6h timepoint. 50 and 500 nM neratinib was more effective at preventing reactivation of ERK1/2 and Akt phosphorylation than either 500 nM lapatinib or tucatinib at 72 h. Conclusion: These data highlight the differences in in vitro efficacy of the three HER2-targeting TKIs currently approved for the treatment of HER2+ breast cancer in the clinic. Neratinib inhibits HER2 and EGFR at lower concentrations and for longer compared to lapatinib and tucatinib, resulting in increased apoptosis induction. This may be due to the irreversible nature of neratinib binding, as opposed to the reversible nature of lapatinib and tucatinib binding. Further comparative studies between neratinib, lapatinib and tucatinib are warranted to explore the impact of varied pre-clinical characteristics on the emergence of therapeutic resistance. Citation Format: Myra E Castel, Neil T Conlon, Naomi Walsh, Irmina Diala, Lisa Eli, John Crown, Denis M Collins. Comparative time course analysis of the effects of neratinib, lapatinib and tucatinib in an in vitro HER2+ breast cancer model [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-36.