Internalization and intracellular processing of an anti-B-cell lymphoma monoclonal antibody, LL2.
The successful clinical experience with antibody LL2 (an IgG2a, anti-B-cell lymphoma antibody) in radioimmunodetection and radioimmunotherapy suggests that this antibody may have potential as a carrier of cytotoxic agents. The internalization, cellular trafficking, and catabolism of this antibody in target human Burkitt lymphoma cells (Raji) were investigated. Internalization of intact antibody as well as of the F(ab')2 and Fab' fragments was detected by an FITC-labeled anti-mouse second antibody probe, and evaluated by fluorescence microscopy. Internalization of intact IgG (or the fragments) was observed as early as 5 min after incubation at 37 degrees C. Initially, the internalized antibodies were present as micro-particles inside the cell membrane, and were translocated to the lysosomal compartment within 2 hr. The anatomic location of the internalized antibody, before translocation to the lysosomal compartment, was deduced by comparing the fluorescence images obtained with the antibody to those obtained with fluorescent probes with known cellular distribution in a co-internalization study. A Golgi-like compartment was found to be involved in the translocation of the antibody. Cellular catabolism of the bound antibody was studied by using 125I-labeled antibody on the target cells. At 21 h, 40% of the radioactivity was released into the supernatant as degraded fragments. The observation suggested that the antibody was degraded mainly in the lysosomes, since the degradation was significantly inhibited in the presence of lysosomal inhibitors such as ammonium chloride or leupeptin. Subcellular fractionation of Raji cells after the binding of 125I-labeled LL2 indicated that the antibody was translocated to lysosomes as evidenced by SDS-PAGE. The rate of internalization (Ke) of LL2, and the re-expression of the antigen were determined. The rapid internalization of LL2 and the re-expression of the antigen suggest that this antibody may have potential as a therapeutic immunoconjugate, since it could deliver a higher accumulation of cytotoxic agents into lymphoma cells.
Shih
L B
LB
Garden State Cancer Center, Center for Molecular Medicine and Immunology, Newark, NJ 07103.
Lu
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Xuan
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Goldenberg
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D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000643:Ammonium Chloride / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D002051:Burkitt Lymphoma / Q000378:metabolism*; D002518:Ceramides / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D016393:Lymphoma, B-Cell / Q000276:immunology*; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002518:Ceramides; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D007976:Leupeptins; C095913:bectumomab; D000643:Ammonium Chloride; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D009327:4-Chloro-7-nitrobenzofurazan; D016650:Fluorescein-5-isothiocyanate; C032854:leupeptin