A novel method for measuring cellular antibody uptake using imaging flow cytometry reveals distinct uptake rates for two different monoclonal antibodies targeting L1

• Verfasst von: Hazin, John [VerfasserIn]
• Verfasst von: Moldenhauer, Gerhard [VerfasserIn]
• Verfasst von: Altevogt, Peter [VerfasserIn]
• Verfasst von: Brady, Nathan [VerfasserIn]
• Titel: A novel method for measuring cellular antibody uptake using imaging flow cytometry reveals distinct uptake rates for two different monoclonal antibodies targeting L1
• Verf.angabe: John Hazin, Gerhard Moldenhauer, Peter Altevogt, Nathan R. Brady
• E-Jahr: 2015
• Jahr: 9 May 2015
• Umfang: 8 S.
• Fussnoten: Gesehen am 16.07.2020
• Titel Quelle: Enthalten in: Journal of immunological methods
• Ort Quelle: Amsterdam [u.a.] : Elsevier Science, 1971
• Jahr Quelle: 2015
• Band/Heft Quelle: 423(2015), Seite 70-77
• ISSN Quelle: 1872-7905
• DOI: doi:10.1016/j.jim.2015.04.024
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: Antibody internalization
• Sach-SW: Endocytosis
• Sach-SW: EpCAM
• Sach-SW: Imaging flow cytometry
• Sach-SW: L1
• Sach-SW: L1CAM
• K10plus-PPN: 1724943332

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.