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The Fate of Antibodies Bound to the Surface of Tumor Cells in Vitro¹

Garden State Cancer Center and Center for Molecular Medicine and Immunology, Newark, New Jersey 07103

The fate of monoclonal antibodies binding to the surface of human tumor cells in vitro was investigated. Seven antibodies, labeled with ¹²⁵I, were tested on four cell lines, which included a melanoma and carcinomas of the ovary, kidney, and lung. The antibodies were selected only by the criterion that they not be rapidly internalized via coated pits, so that they would be representative of most antibodies reacting with cell surface antigens. After allowing binding during a 2-h incubation, unbound antibody was removed, and the release of intact or degraded antibody in the supernatant was monitored. The data demonstrate that most bound antibody was gradually degraded and released from the cell over a 2–3-day period, probably via internalization, while only a small fraction, <20% for most antibodies, appeared to dissociate intact. One exceptional antibody, MW207, dissociated largely intact. The release of intact antibody was virtually complete within 4 h, and radioactivity released after this time was predominantly in degraded form. These results demonstrate that antibody binding to the surface of viable cells must in general be considered irreversible, and hence the concept of affinity is not applicable. Since an Fab fragment of one of the antibodies dissociated rapidly, such irreversible binding appears to require bivalent attachment. Another conclusion of this study is that most antibodies binding to the cell surface are gradually internalized, which we suggest is due to the normal turnover of cell surface constituents via non-clathrin-dependent endocytosis. Several experimental approaches indicated that a large fraction of antibody retained by the cells, for at least 2 days after binding, was present at the cell surface.

¹ Supported in part by USPHS Grants CA48779, CA39841, and RR05903 from the NIH.

² To whom requests for reprints should be addressed, at Garden State Cancer Center and Center for Molecular Medicine and Immunology, 1 Bruce Street, Newark, NJ 07103.

Received 7/23/91. Accepted 10/24/91.

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