Abstract PS10-08: Tucatinib potentiates the activity of the antibody-drug conjugate T-DM1 in preclinical models of HER2-positive breast cancer
Authors: Kulukian Anita , Taylor Janelle , Jain Nishi , Olson Devra , Zaval Margo , Thurman Robert , Hengel Shawna , Farr Lauren , Pires Thomas , Peterson Scott R. 
Published: February 2021

Abstract
Background: Tucatinib is an orally administered, reversible, highly specific HER2 tyrosine kinase inhibitor recently approved by the FDA in combination with trastuzumab and capecitabine for adult patients with advanced unresectable or metastatic HER2-positive breast cancer (mBC), including patients with brain metastases, that have failed at least one anti-HER2 regimen in the metastatic setting. In a phase IB clinical trial, tucatinib in combination with the HER2-targeted antibody-drug conjugate (ADC) ado-trastuzumab emtansine (T-DM1) was well tolerated and demonstrated activity in heavily pre-pretreated patients with HER2-positive mBC (NCT01983501; Borges VF et al., 2018). We previously presented preclinical data that tucatinib increases the activity of trastuzumab-derived ADCs in HER2-positive breast cancer models. Here, we provide mechanistic insight that tucatinib potentiates the activity of T-DM1 by modulating HER2 protein dynamics and facilitating increased cytotoxic maytansinoid drug delivery. Methods: To assess changes to HER2 protein levels upon treatment with tucatinib, HER2-amplified breast cancer cell lines were analyzed by Western blot and quantitative FACS (qFACS). To probe the dynamics of HER2 at the cell surface upon binding to antibody therapeutics, SK-BR-3 cells were incubated with fluorescently labeled trastuzumab to mark HER2 at the cell surface. Cells were imaged over 72 hours to observe the internalization of surface-bound antibody. Concurrent experiments were conducted with trastuzumab labeled with QF01, a quenched fluor which fluoresces only upon lysosomal processing and can serve as a proxy for antibody catabolism. To directly measure the rates of ADC catabolism, lysates were generated from BT-474 cells treated with T-DM1 in the presence or absence of tucatinib over a 72 hour time course, and were analyzed by mass spectrometry for the T-DM1 adduct, Lys-MCC-DM1. Results: In HER2-amplified breast cancer cell lines, treatment with tucatinib increased overall and cell membrane-localized HER2 levels. As demonstrated by internalization assays, tucatinib had an initial effect that increased the dwell time of HER2 at the cell surface of SK-BR-3 cells. At later timepoints, HER2 bound to trastuzumab was internalized and directed towards lysosomes. These data were supported by parallel intracellular Lys-MCC-DM1 measurements, which demonstrated increased concentration of the adduct when TDM-1 was administered in combination with tucatinib. These data provide a mechanistic rationale as to why the co-administration of tucatinib with T-DM1 in vitro was synergistic by isobologram analysis, and why the combination of tucatinib with T-DM1 was more effective in vivo than either single agent alone in BT-474 xenografts and in PDX models tested, producing a higher proportion of partial or complete tumor regressions. Conclusions: The described preclinical in vitro and in vivo data of simultaneous dual HER2 inhibition with tucatinib and T-DM1, along with the results of the phase IB/2 clinical trial demonstrating preliminary safety and efficacy of the combination, warrant further clinical development of tucatinib in combination with T-DM1. These results also support the evaluation of tucatinib in combination with other HER2-targeted ADCs in patients with HER2-positive mBC.
Citation Format: Anita Kulukian, Janelle Taylor, Nishi Jain, Devra Olson, Margo Zaval, Robert Thurman, Shawna Hengel, Lauren Farr, Thomas Pires, Scott R. Peterson. Tucatinib potentiates the activity of the antibody-drug conjugate T-DM1 in preclinical models of HER2-positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-08.