Internalization and degradation of monoclonal antibody chCE7 by human neuroblastoma cells.
Internalization and cellular degradation of a chimeric monoclonal anti-neuroblastoma antibody (MAb chCE7) is described. Immunofluorescence localization showed temperature-dependent redistribution of MAb chCE7 from the cell surface at 0 degrees C to vesicular structures after heating to 37 degrees C. SKN-AS cells which were pulse-labelled at 0 degrees C with radioiodinated chCE7 released 50% of the initial cell-bound radioactivity into the medium after 20 hr at 37 degrees C. Low-molecular-weight radioactivity in the medium was identified as iodotyrosine and the major intracellular degradation product was found to be an antibody fragment of 43 kDa. Degradation was blocked by chloroquine, an inhibitor of lysosomal function. MAb chCE7 binds to a carbohydrate epitope, as treatment with tunicamycin abolishes cell binding. In adherent cells in culture, inhibition of protein synthesis by cycloheximide affected cell-surface binding sites for chCE7 in a biphasic manner, with an initial increase followed by long-term decrease. Expression of binding sites was also found to be inhibited by sodium azide, by the lysosomotropic drug chloroquine and by Brefeldin A, a drug which prevents export of newly synthetized proteins to the cell surface. When cells were treated with high doses of chCE7 up to 24 hr and cell-surface binding sites were then measured after an acidic buffer wash, no loss of surface binding sites was found, indicating that pretreatment with MAb chCE7 does not induce down-regulation of its binding sites.
Novak-Hofer
I
I
Radiopharmacy Division, Paul Scherrer Institute, Villigen-PSI, Switzerland.
Amstutz
H P
HP
Morgenthaler
J J
JJ
Schubiger
P A
PA
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D009447:Neuroblastoma / Q000276:immunology / Q000378:metabolism*; D013696:Temperature
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D007457:Iodine Radioisotopes