Tumor Cells Chronically Treated with a Trastuzumab–Maytansinoid Antibody–Drug Conjugate Develop Varied Resistance Mechanisms but Respond to Alternate Treatments

Abstract

Antibody-drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab-maytansinoid ADC (TM-ADC) at IC 80 concentrations followed by recovery. The resistant cells, 361-TM and JIMT1-TM, were characterized by cytotoxicity, proteomic, transcriptional, and other profiling. Approximately 250-fold resistance to TM-ADC developed in 361-TM cells, and cross-resistance was observed to other non-cleavable-linked ADCs. Strikingly, these 361-TM cells retained sensitivity to ADCs containing cleavable mcValCitPABC-linked auristatins. In JIMT1-TM cells, 16-fold resistance to TM-ADC developed, with cross-resistance to other trastuzumab-ADCs. Both 361-TM and JIMT1-TM cells showed minimal resistance to unconjugated mertansine (DM1) and other chemotherapeutics. Proteomics and immunoblots detected increased ABCC1 (MRP1) drug efflux protein in 361-TM cells, and decreased Her2 (ErbB2) in JIMT1-TM cells. Proteomics also showed alterations in various pathways upon chronic exposure to the drug in both cell models. Tumors derived from 361-TM cells grew in mice and were refractory to TM-ADC compared with parental cells. Hence, acquired resistance to trastuzumab-maytansinoid ADC was generated in cultured cancer cells by chronic drug treatment, and either increased ABCC1 protein or reduced Her2 antigen were primary mediators of resistance. These ADCresistant cell models retain sensitivity to other ADCs or standardof-care chemotherapeutics, suggesting that alternate therapies may overcome acquired ADC resistance.

Caveolae-Mediated Endocytosis as a Novel Mechanism of Resistance to Trastuzumab Emtansine (T-DM1)

“…ADCs and chemical structures of disclosed payloads described herein were prepared as previously described (13). The syntheses and structures of the novel auristatin linker-payloads as well as preparation and characterization of ADCs presented herein are described in the Supplementary Methods.…”

“…Cellular responses to ADCs and free drugs were measured with a cytotoxicity assay as previously described (13). Briefly, cells were seeded into 96-well plates and treated with varying concentrations of ADCs or free drugs for 96 hours.…”

“…Proteomic profiling of membrane fractions was performed and analyzed as previously described (13). Whole-cell lysates were prepared in RIPA buffer supplemented with protease inhibitor cocktail (Sigma).…”

“…Previous reports of T-DM1 resistance in other cell models have implicated ATP-binding cassette (ABC) transporter protein overexpression (e.g., ABCB1/MDR1, ABCC1/MRP1) or target antigen downregulation as key drivers of T-DM1 resistance (13,14). However, proteomics and immunoblots demonstrate that N87-TM cells do not overexpress MDR1 (Fig.…”

“…Although data are still emerging from T-DM1-treated patient samples, multiple pre-clinical models of T-DM1 resistance have been generated to characterize the changes at the tumor cell level that may mediate clinical resistance to T-DM1. Although the models of T-DM1 resistance in these studies were generated with different dosing schemas, the following are mechanisms responsible for T-DM1 resistance seen across multiple models: (i) decreased HER2 expression (13,14), (ii) increased expression and activity of drug efflux proteins (13,14), and (iii) increased heregulin expression (15).…”