Dynamics of Endocytosis and Degradation of Antibody-Drug Conjugate T-DM1 in HER2 Positive Cancer Cells

Abstract

Purpose

T-DM1 is an antibody–drug conjugate (ADC) consisting of trastuzumab and DM1 linked together. T-DM1 binds to human epidermal growth factor receptor-2 (HER2) in tumors and then triggers the endocytosis of T-DM1 and release of payload. Therefore, endocytosis efficacy is considered as a critical step for the initiation of T-DM1 therapy; however, the endocytosis mechanism of T-DM1 remains poorly understood. Meanwhile, HER2 is regarded as an internalization-resistant receptor, which hinders the endocytosis and effectiveness of T-DM1. The present study is to explore the T-DM1 endocytosis pathway, which may provide insights into the internalization mechanism of ADCs and help to improve efficacy.

Methods

Confocal microscopy and flow cytometry were used to analyse T-DM1 intracellular trafficking and endocytosis efficiency, while Western blot assay was performed to detect T-DM1 degradation.

Results

We found that intracellular T-DM1 was increased to 50% within 12 h. T-DM1 was colocalized with cholera toxin B (CTxB), a lipid raft marker, within 2 h and then degraded in lysosome. Upon overexpression of caveolin-1 (CAV-1) and utilization of caveolae/lipid-raft disruptors, we found that temporal CAV-1 upregulation significantly facilitated T-DM1 endocytosis and degradation, whereas nystatin and lovastatin disrupted caveolae/lipid-raft structure and inhibited T-DM1 degradation. We demonstrate that T-DM1 internalizes through the lipid raft-mediated endocytosis in a CAV-1 dependent manner, rather than through the clathrin-mediated endocytosis in HER2-positive cancer cells.

Conclusion

Our findings suggest that modulation of the caveolae/lipid-raft mediated endocytosis may be a possible option for improving the clinical therapeutic effect of T-DM1 because it plays a key role in regulating T-DM1 internalization.

Keywords:

• antibody–drug conjugate
• T-DM1
• endocytosis
• lipid rafts
• caveolae
• caveolin-1

Acknowledgments

This work was supported by the National Natural Science Foundation of China [grant no. 81872784]. We wish to thank for technique supports in Confocal Microscopy and Flow Cytometry from Core Facilities of Zhejiang University School of Medicine.

Abbreviations

ADC, antibody–drug conjugate; CAV-1, caveolin-1; CME, clathrin-mediated endocytosis; CPZ, chlorpromazine; CQ, chloroquine; CTxB, cholera toxin B; HER2, human epidermal growth factor receptor-2; T-DM1, Trastuzumab emtansine.

Disclosure

The authors report no conflicts of interest in this work.