In Vitro Characterization of Internalization Pathways and Cytotoxic Activity of Anti-HSPG2 Antibody-Drug Conjugates in MDA-MB-231-LM2 Cells.

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.