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Introducing a positive charge on the antibody is one proposed strategy to enhance tumor distribution and efficacy of ADC. Accordingly, this study evaluates the pharmacokinetics (PK) and pharmacology of an ADC developed using a positively charged (+5) version of anti-HER2 antibody trastuzumab conjugated with vc-MMAE linker-payload. Methods: A positively charged variant of trastuzumab was generated and conjugated to vc-MMAE. In vitro cytotoxicity assays were performed in cell lines with varying HER2 expression levels: N87 (high), MCF-7 (low), and MDA-MB-468 (non-expressing). In vivo biodistribution of wild-type (WT) and positively charged (+5) ADC was investigated in plasma, tumors, liver, and spleen. A pilot efficacy and toxicity study was also conducted in N87 tumor-bearing mice. Results: The charged ADC showed differential potency and PK behavior compared to the WT ADC. The charged ADC had similar potency in N87 cells but demonstrated ~20-fold and ~60-fold higher potency in MCF-7 and MDA-MB-468 cells. Plasma exposures of all the analytes were found to be reduced following the administration of charged ADC. However, total antibody exposure was found to increase in liver, spleen, and low antigen-expressing MCF-7 tumors. Tumor payload exposures were found to be significantly reduced for the charged ADCs, but liver and spleen displayed higher peak concentrations and increased tissue-to-plasma exposure ratios for the payload, suggesting preferential distribution of ADC with high drug\u2013antibody ratio (DAR) to liver and spleen. Consistent with reduced tumor exposures, charged ADC showed lower efficacy in N87 tumor-bearing mice. No overt toxicity was observed for the charged ADC. Conclusions: Our findings suggest that while positively charged ADCs may be more potent in vitro, their efficacy in vivo may be compromised due to altered PK behavior. Thus, introducing a positive charge into the antibody framework may not be a viable strategy for improving the therapeutic potential of ADCs.<\/jats:p>","DOI":"10.3390\/pharmaceutics17030377","type":"journal-article","created":{"date-parts":[[2025,3,17]],"date-time":"2025-03-17T07:49:57Z","timestamp":1742197797000},"page":"377","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["PK\/PD of Positively Charged ADC in Mice"],"prefix":"10.3390","volume":"17","author":[{"given":"Hsuan-Ping","family":"Chang","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY 14214, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huyen Khanh","family":"Le","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY 14214, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shufang","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY 14214, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0723-6206","authenticated-orcid":false,"given":"Dhaval K.","family":"Shah","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY 14214, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"4677","DOI":"10.1002\/cam4.4052","article-title":"The promising role of antibody drug conjugate in cancer therapy: Combining targeting ability with cytotoxicity effectively","volume":"10","author":"Li","year":"2021","journal-title":"Cancer Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1186\/s12943-024-01963-7","article-title":"A review of the clinical efficacy of FDA-approved antibody\u2012drug conjugates in human cancers","volume":"23","author":"Liu","year":"2024","journal-title":"Mol. 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