{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,23]],"date-time":"2026-05-23T05:05:13Z","timestamp":1779512713858,"version":"3.53.1"},"reference-count":31,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T00:00:00Z","timestamp":1779148800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Background\/objectives: This study presents a mechanistic assessment of an anti-HSPG2 monoclonal antibody (AM6) as an antibody\u2013drug 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.<\/jats:p>","DOI":"10.3390\/cancers18101638","type":"journal-article","created":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T13:11:31Z","timestamp":1779196291000},"page":"1638","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["In Vitro Characterization of Internalization Pathways and Cytotoxic Activity of Anti-HSPG2 Antibody\u2013Drug Conjugates in MDA-MB-231-LM2 Cells"],"prefix":"10.3390","volume":"18","author":[{"given":"Zekun","family":"Shao","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, Philadelphia, PA 19140, USA"},{"name":"Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA 98195, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lauren","family":"Morelli","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, Philadelphia, PA 19140, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin E.","family":"Blass","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, Philadelphia, PA 19140, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-9674-0039","authenticated-orcid":false,"given":"Andrey","family":"Efimov","sequence":"additional","affiliation":[{"name":"Fox Chase Comprehensive Cancer Institute, Temple University, Philadelphia, PA 19111, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8656-2244","authenticated-orcid":false,"given":"Jayanth","family":"Panyam","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutics, College of Pharmacy, University of Washington, Seattle, WA 98195, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1938","DOI":"10.1056\/NEJMra1001389","article-title":"Triple-negative breast cancer","volume":"363","author":"Foulkes","year":"2010","journal-title":"N. 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