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.

Exploration of the antibody–drug conjugate clinical landscape

“…A favorable example of a target that is significantly overexpressed in tumor tissues relative to normal tissues is the HER2/neu antigen that is expressed at lower levels on a subset of normal cells, but expressed at hundreds of thousands to over a million copies on HER2+ cancer cells. 5 Indeed, ADCs targeting the HER2 antigen have demonstrated robust internalization into HER2-targeted tumor cells with efficient payload delivery 6 , 7 that has translated to clinical benefit and ultimate drug approval. 8 , 9 In contrast, ADCs targeting tumor antigens with heterogeneous/low target antigen expression, such as the prolactin receptor with antigen densities of thousands to tens of thousands of molecules/cell, 10 failed to demonstrate clinical responses at the biologic doses tested and were subsequently terminated from future clinical development.…”

Computational design of fusion proteins against ErbB2-amplified tumors inspired by ricin toxin

“…MD results indicated that ricin A of CPs 2, 5, 7, 8, and 10 may most likely retain its inhibitory activity. Using molecular docking, we aimed to examine whether the selected CPs were capable of binding to trastuzumab-binding pocket on ErbB-2 since this region internalizes trastuzumab upon its binding (Austin et al, 2004) in a Caveolae/Lipid-Raft Mediated mechanism (Liang et al, 2021). The last frame of each MD trajectory entered the docking step.…”

How can we use the endocytosis pathways to design nanoparticle drug-delivery vehicles to target cancer cells over healthy cells?

“…143,144 HER2 has been linked to the activation of both clathrin-mediated and caveolae-mediated endocytosis uptakes. [145][146][147] Hence why it has been a popular anti-cancer target as these endocytosis pathways are common in most cancer cells.…”