Abstract 618: Role of multidrug resistance transporters in the biological response to trastuzumab-cytotoxic drug conjugates

Abstract

Abstract Trastuzumab-DM1 (T-DM1, TMAb-mcc-DM1, trastuzumab emtansine) is an antibody-cytotoxic drug conjugate that is composed of the maytansine derivative DM1 directly coupled, via a thioether SMCC linker, to trastuzumab. T-DM1 is currently undergoing clinical evaluation in metastatic HER2-positive breast cancer patients. T-DM1 has been shown to retain all of the mechanisms of action of trastuzumab and to deliver DM1 to HER2-overexpressing tumor cells. In the present study, we investigated potential mechanisms of resistance to T-DM1 and other trastuzumab conjugates, including a different class of anti-microtubule drugs, the auristatins (MMAE and MMAF). Expression of selected multi-drug resistance (MDR) transporters MDR1, MRP1, MRP3 and BCRP was determined by qRT-PCR (Taqman). MDR1 was expressed at moderate levels by only one line, while expression of MRP3 occurred in several HER2-overexpressing cell lines. Cell viability assays with either free drugs (DM1 or MMAE) or the respective trastuzumab conjugates showed that all lines were sensitive to treatment with these agents. The NCI-AdrRes line, which expresses high levels of MDR1, was transfected to overexpress HER2 and used as a positive control. This line was resistant to treatment with DM1 or MMAE, consistent with previous reports that these drugs are substrates for MDR1. Additional studies were performed with the clinical conjugate, T-DM1, on the HER2-amplified breast cancer line SK-BR-3 transfected to express either MDR1, MRP1, MRP3 or BCRP, focusing on the clinical conjugate, T-DM1. In cell proliferation assays, both NCI-AdrRes/HER2 and SK-BR-3/MDR1 were resistant to free DM1, and the addition of XR9051 (a specific MDR1 inhibitor) reversed this resistance. Although the sensitivity to T-DM1 for both cell lines was restored by the addition of XR9051, NCI-AdrRes/HER2 showed more resistance to T-DM1 in the absence of XR9051. SK-BR-3 cells expressing MRP1 or BCRP maintained sensitivity to free and conjugated drugs, while cells expressing MRP3 showed a very modest induction of resistance only at high drug concentrations. The effects of linker stability were also investigated by comparing trastuzumab conjugates comprised of stable vs. cleavable (reducible or protease-sensitive) linkers. MDR1 expressing cells showed relatively more resistance to conjugates with cleavable linkers, presumably due to efflux of free drug upon release after linker cleavage. Together, these data show that, of the MDR transporters studied, only MDR1 confers high level resistance to trastuzumab-cytotoxic drug conjugates comprised of DM1 or auristatins, and that linking the drug through a stable linker may allow evasion of drug efflux through MDR-mediated mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 618.