title: A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies.
abstract: Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2'-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell-based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2'-deoxyuridine (EdU) and 4',6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs.
authors.affiliation: 1 Department of Biology II, LMU Munich, Planegg-Martinsried, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Stengl
authors.affiliation: 1 Department of Biology II, LMU Munich, Planegg-Martinsried, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Hörl
authors.affiliation: 1 Department of Biology II, LMU Munich, Planegg-Martinsried, Germany.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.affiliation: 1 Department of Biology II, LMU Munich, Planegg-Martinsried, Germany.
authors.forename: Jonas
authors.initials: J
authors.lastname: Helma
mesh_terms: D000074322:Antineoplastic Agents, Immunological / Q000494:pharmacology*; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000737:chemistry; D005260:Female; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D007211:Indoles / Q000737:chemistry; D042361:Mammary Glands, Human / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D000068878:Trastuzumab / Q000494:pharmacology*
chemical_list: D000074322:Antineoplastic Agents, Immunological; D014408:Biomarkers, Tumor; D003857:Deoxyuridine; D007211:Indoles; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; C007293:DAPI; C508053:ERBB2 protein, human; C031086:5-ethynyl-2'-deoxyuridine
keywords: EdU; cell-based assays; high-content screening; proliferation; therapeutic antibodies