This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
In Vitro Characterization of Internalization Pathways and Cytotoxic Activity of Anti-HSPG2 Antibody–Drug Conjugates in MDA-MB-231-LM2 Cells
1
Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, Philadelphia, PA 19140, USA
2
Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA
3
Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA 98195, USA
4
Fox Chase Comprehensive Cancer Institute, Temple University, Philadelphia, PA 19111, USA
5
Department of Pharmaceutics, College of Pharmacy, University of Washington, Seattle, WA 98195, USA
*
Author to whom correspondence should be addressed.
Cancers 2026, 18(10), 1638; https://doi.org/10.3390/cancers18101638
Submission received: 13 April 2026
/
Revised: 13 May 2026
/
Accepted: 15 May 2026
/
Published: 19 May 2026
(This article belongs to the Special Issue Advances in Antibody–Drug Conjugates (ADCs) in Cancers)
Simple Summary
Antibody–drug conjugates are a promising type of targeted cancer therapy designed to deliver potent drugs directly to tumor cells while minimizing harm to healthy tissue. However, the effectiveness of a conjugate depends on how well it enters cancer cells and releases its drug payload inside them. In this study, we examine an antibody called AM6 that targets HSPG2, a protein commonly found on certain cancer cells, to better understand how cells take it up and how different drug-attachment strategies influence its activity. Using advanced imaging techniques, we tracked how AM6 moves inside cancer cells and tested several drug-linker designs. We found that certain drug combinations led to stronger cancer cell killing than others. These results provide practical guidance for designing more effective ADCs and highlight important considerations for future laboratory and preclinical studies in the field.
Abstract
Background/objectives: This study presents a mechanistic assessment of an anti-HSPG2 monoclonal antibody (AM6) as an antibody–drug conjugate (ADC) carrier in vitro. Methods: Using live-cell confocal imaging with pathway inhibitors, we qualitatively characterized AM6 internalization and trafficking and compared linker/payload configurations for intracellular delivery and in vitro cytotoxicity. Results: AM6 exhibited rapid cellular entry in MDA-MB-231-LM2 cells, with contributions from clathrin-mediated endocytosis and macropinocytosis, followed by accumulation in endo-lysosomal compartments. Consistent with these trafficking observations, AM6 ADCs bearing cleavable linkers and a potent payload (MMAE) produced more pronounced antiproliferative effects in MDA-MB-231-LM2 and other HSPG2-positive tumor cells than non-cleavable constructs, whereas doxorubicin-based ADCs showed limited activity and greater aggregation risk. Conclusions: Overall, the data inform linker/payload selection and highlight considerations for future work, including quantitative internalization, antigen-negative or knockdown controls, and in vivo pharmacology.
Keywords:
ADC; linker chemistry; cellular internalization; cytotoxicity
