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Endocytosis of Immunotoxin-791T/36-RTA by Tumor Cells in Relation to Its Cytotoxic Action¹

Cancer Research Campaign Laboratories, University of Nottingham, Nottingham NG7 2RD, United Kingdom [V. S. B., I. Z. A. P., D. S. W. H., M. R. P., M. J. E., M. C. G., N. B., R. A. R., R. W. B.], and Xoma Corporation, Berkeley, California 94710 [V. S. B., S. C.]

Ricin A chain immunotoxin constructed with monoclonal antibody 791T/36, which recognizes a tumor associated glycoprotein M_r 72,000 antigen present on sarcomas and colon and ovarian cancer cells, is cytotoxic for cell lines from tumors expressing this antigen. Incubation of sarcoma 791T cells with immunotoxin for only 5 min is sufficient to produce >95% inhibition of tumor cell growth. Papain treatment of these cells to remove immunotoxin from the cell surface indicated that the cell surface acts as a reservoir for continued internalization of immunotoxin over several hours, but even so, 50% inhibition of cell survival was produced over a 2- to 3-h period.

Analysis of the rate of endocytosis demonstrated that 30–50% of cell bound immunotoxin was internalized over a 180-min period. This was primarily dictated by the antibody moiety, regardless of the degree of conjugation to ricin A chain. This rate is much slower than that of other cell surface ligands such as transferrin. Cell cytosol acidification experiments were performed to determine whether this immunotoxin was internalized by clathrin coated pits, which is relatively rapid, or by smooth pits, which is slower, and the results indicated the latter mechanism is almost exclusively used. Intracellular trafficking of antibody 791T/36, conjugated to human serum albumin-tetramethylrhodamine was investigated by flow cytometry. The movement of the conjugate into the lysosomal compartment was delayed so that degradation products were only detected after a lag phase of 30–60 min. The lack of potentiator dependence of 791T/36 immunotoxin is in keeping with these findings.

¹ These studies were supported by the Xoma Corporation and by the Cancer Research Campaign, United Kingdom.

² To whom requests for reprints should be addressed, at Cancer Research Campaign Laboratory, University of Nottingham, Nottingham NG7 2RD, U.K.

Received 10/15/90. Accepted 1/31/91.

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