title: A novel method for measuring cellular antibody uptake using imaging flow cytometry reveals distinct uptake rates for two different monoclonal antibodies targeting L1.
abstract: Monoclonal antibodies (mAbs) have emerged as a promising tool for cancer therapy. Differing approaches utilize mAbs to either deliver a drug to the tumor cells or to modulate the host's immune system to mediate tumor kill. The rate by which a therapeutic antibody is being internalized by tumor cells is a decisive feature for choosing the appropriate treatment strategy. We herein present a novel method to effectively quantitate antibody uptake of tumor cells by using image-based flow cytometry, which combines image analysis with high throughput of sample numbers and sample size. The use of this method is established by determining uptake rate of an anti-EpCAM antibody (HEA125), from single cell measurements of plasma membrane versus internalized antibody, in conjunction with inhibitors of endocytosis. The method is then applied to two mAbs (L1-9.3, L1-OV52.24) targeting the neural cell adhesion molecule L1 (L1CAM) at two different epitopes. Based on median cell population responses, we find that mAb L1-OV52.24 is rapidly internalized by the ovarian carcinoma cell line SKOV3ip while L1 mAb 9.3 is mainly retained at the cell surface. These findings suggest the L1 mAb OV52.24 as a candidate to be further developed for drug-delivery to cancer cells, while L1-9.3 may be optimized to tag the tumor cells and stimulate immunogenic cancer cell killing. Furthermore, when analyzing cell-to-cell variability, we observed L1 mAb OV52.24 rapidly transition into a subpopulation with high-internalization capacity. In summary, this novel high-content method for measuring antibody internalization rate provides a high level of accuracy and sensitivity for cell population measurements and reveals further biologically relevant information when taking into account cellular heterogeneity.
authors.affiliation: Department of Translational Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany; Department of Surgery, Medical Faculty, University of Heidelberg, Heidelberg, Germany; Systems Biology of Cell Death Mechanisms, German Cancer Research Center (DKFZ), Heidelberg, Germany. Electronic address: j.hazin@dkfz.de.
authors.forename: John
authors.initials: J
authors.lastname: Hazin
authors.affiliation: Department of Translational Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Moldenhauer
authors.affiliation: Department of Translational Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Altevogt
authors.affiliation: Department of Surgery, Medical Faculty, University of Heidelberg, Heidelberg, Germany; Systems Biology of Cell Death Mechanisms, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Brady
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology*; D002277:Carcinoma / Q000378:metabolism; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D019044:Image Cytometry / Q000379:methods; D007136:Immunoglobulins / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; C403514:CADM1 protein, human; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D007136:Immunoglobulins
keywords: Antibody internalization; Endocytosis; EpCAM; Imaging flow cytometry; L1; L1CAM