INTERNALIZATION AND INTRACELLULAR PROCESSING OF AN ANTI B-CELL LYMPHOMA MONOCLONAL-ANTIBODY, LL2

The successful clinical experience with antibody LL2 (an IgG(2a), anti -B-cell lymphoma antibody) in radioimmunodetection and radioimmunother apy suggests that this antibody may have potential as a carrier of cyt otoxic agents. The internalization, cellular trafficking, and cataboli sm 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. Inter nalization of intact IgG (or the fragments) was observed as early as 5 min after incubation at 37 degrees C. Initially, the internalized ant ibodies were present as micro-particles inside the cell membrane, and were translocated to the lysosomal compartment within 2 hr. The anatom ic location of the internalized antibody, before translocation to the lysosomal compartment, was deduced by comparing the fluorescence image s obtained with the antibody to those obtained with fluorescent probes with known cellular distribution in a co-internalization study. A Gol gi-like compartment was found to be involved in the translocation of t he antibody. Cellular catabolism of the bound antibody was studied by using I-125-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 th e lysosomes, since the degradation was significantly inhibited in the presence of lysosomal inhibitors such as ammonium chloride or leupepti n. Subcellular fractionation of Raji cells after the binding of I-125- labeled LL2 indicated that the antibody was translocated to lysosomes as evidenced by SDS-PAGE. The rate of internalization (K-e) of LL2, an d the re-expression of the antigen were determined. The rapid internal ization 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. (C) 1994 Wiley-Liss, Inc.