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000126676 0247_ $$2doi$$a10.1016/j.jim.2015.04.024
000126676 0247_ $$2pmid$$apmid:25967950
000126676 0247_ $$2ISSN$$a0022-1759
000126676 0247_ $$2ISSN$$a1872-7905
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000126676 037__ $$aDKFZ-2017-02704
000126676 041__ $$aeng
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000126676 1001_ $$0P:(DE-HGF)0$$aHazin, John$$b0$$eFirst author
000126676 245__ $$aA novel method for measuring cellular antibody uptake using imaging flow cytometry reveals distinct uptake rates for two different monoclonal antibodies targeting L1.
000126676 260__ $$aAmsterdam [u.a.]$$bElsevier Science$$c2015
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000126676 520__ $$aMonoclonal antibodies (mAbs) have emerged as a promising tool for cancer therapy. Differing approaches utilize mAbs to either deliver a drug to the tumor cells or to modulate the host's immune system to mediate tumor kill. The rate by which a therapeutic antibody is being internalized by tumor cells is a decisive feature for choosing the appropriate treatment strategy. We herein present a novel method to effectively quantitate antibody uptake of tumor cells by using image-based flow cytometry, which combines image analysis with high throughput of sample numbers and sample size. The use of this method is established by determining uptake rate of an anti-EpCAM antibody (HEA125), from single cell measurements of plasma membrane versus internalized antibody, in conjunction with inhibitors of endocytosis. The method is then applied to two mAbs (L1-9.3, L1-OV52.24) targeting the neural cell adhesion molecule L1 (L1CAM) at two different epitopes. Based on median cell population responses, we find that mAb L1-OV52.24 is rapidly internalized by the ovarian carcinoma cell line SKOV3ip while L1 mAb 9.3 is mainly retained at the cell surface. These findings suggest the L1 mAb OV52.24 as a candidate to be further developed for drug-delivery to cancer cells, while L1-9.3 may be optimized to tag the tumor cells and stimulate immunogenic cancer cell killing. Furthermore, when analyzing cell-to-cell variability, we observed L1 mAb OV52.24 rapidly transition into a subpopulation with high-internalization capacity. In summary, this novel high-content method for measuring antibody internalization rate provides a high level of accuracy and sensitivity for cell population measurements and reveals further biologically relevant information when taking into account cellular heterogeneity.
000126676 536__ $$0G:(DE-HGF)POF3-312$$a312 - Functional and structural genomics (POF3-312)$$cPOF3-312$$fPOF III$$x0
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000126676 650_7 $$2NLM Chemicals$$aAntibodies, Monoclonal
000126676 650_7 $$2NLM Chemicals$$aAntigens, Neoplasm
000126676 650_7 $$2NLM Chemicals$$aCADM1 protein, human
000126676 650_7 $$2NLM Chemicals$$aCell Adhesion Molecules
000126676 650_7 $$2NLM Chemicals$$aEPCAM protein, human
000126676 650_7 $$2NLM Chemicals$$aEpithelial Cell Adhesion Molecule
000126676 650_7 $$2NLM Chemicals$$aImmunoglobulins
000126676 7001_ $$0P:(DE-HGF)0$$aMoldenhauer, Gerhard$$b1
000126676 7001_ $$0P:(DE-He78)e6057131c71c90a6fcf61035f12b46c3$$aAltevogt, Peter$$b2$$udkfz
000126676 7001_ $$0P:(DE-He78)5bf984e94f0a31773a103cd293e01f92$$aBrady, Nathan R$$b3$$eLast author$$udkfz
000126676 773__ $$0PERI:(DE-600)1500495-8$$a10.1016/j.jim.2015.04.024$$gVol. 423, p. 70 - 77$$p70 - 77$$tJournal of immunological methods$$v423$$x0022-1759$$y2015
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