Internalization of a monoclonal antibody recognizing carcinoembryonic antigen (CEA) by human cancer cell lines

Tsaltas, Georgia-Zetta H. (1994) Internalization of a monoclonal antibody recognizing carcinoembryonic antigen (CEA) by human cancer cell lines. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Monoclonal antibody (Mab) internalization by cancer cells has been recently gaining increasing recognition as one of the important factors affecting the action of Mabs or immunoconjugates (ICs) on tumour sites. This project addresses the underexplored subject of internalization, by comparing existing internalization assays in terms of accuracy and consistency, as well as by developing more rapid and concise methods for the detection of internalized antibody. A number of in vitro techniques for investigating internalization are evaluated, using a model which consists of a well characterized anticarcinoembryonic antigen (anti-CEA) Mab and a number of CEA expressing human cancer cell lines. Employing two alternative radiolabelling assays, evidence for internalization of an anti-CEA Mab by a CEA-positive colorectal cancer cell line (LS174T) was obtained throughout the time intervals examined (5 min to 150 min.). A widely employed internalization assay involving the use of a low pH buffer for the dissociation of surface antigen-antibody bonds, has been thoroughly analyzed and shown not to fulfill its alleged role, thereby introducing inaccuracies in the experimental method. Electronmicroscopy employing horseradish-peroxidase labelled anti-CEA Mab, permitted the direct visualization of anti-CEA Mab related staining in intracellular compartments of a high CEA-expressor human colorectal cell line (SKCO1). SDS/PAGE analysis of samples derived from cytosolic and membrane components of solubilized cells from lung and colonic cancer cell lines, provided evidence for non-degraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 minutes. Internalized anti-CEA was detected in all CEA expressing cell lines (LS174T, SKCO1, BENN) but not in the case of a very low CEA expressor line (COLO 320). When the last method was compared to a newly developed internalization assay involving flow cytometry, results were very similar for all the above cell lines. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a fast and uncomplicated alternative to the internalization assays used at present. Finally, the question of the endocytic route followed by CEA-anti-CEA complexes is addressed through blocking clathrin-mediated endocytosis in the cell lines examined. Preliminary results indicate that such complexes may be internalized only partially by clathrin-coated vesicles, with an alternative uptake-endocytic mechanism also at work.

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