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Comparative Cellular Catabolism and Retention of Astatine-, Bismuth-, and Lead-Radiolabeled Internalizing Monoclonal Antibody

Department of Nuclear Medicine and PET Department, Warren G. Magnuson Clinical Center; and Metabolism and Radiation Oncology Branches, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

View larger version (26K): [in a new window]   FIGURE 1. Retention and release of 125I-, 211At-, 111In-, 205,6Bi-, and 203Pb-labeled T101 by PBMNC were determined. PBMNC (106) were incubated with 100 ng trace-labeled T101 at 4°C for 1 h and then washed, placed in fresh culture medium, and incubated at 37°C for 0, 1, 2, 4, and 8 h before quantifying amounts of cell-associated radioactivity and amount released into supernatant. Supernatant radioactivity was fractionated into methanol-precipitable noncatabolized component and methanol-soluble catabolized fraction. SD bars are plotted for replicates but are too small to be visualized for most time points.

View larger version (24K): [in a new window]   FIGURE 2. Retention and release of various - and -emitter–labeled T101s by MOLT-4 cell line were determined as described for Figure 1.

View larger version (24K): [in a new window]   FIGURE 3. Percentage (mean ± SD) of acid-resistant to total cell-bound radioactivity for radiolabeled T101 by PBMNC. Labeled T101 was allowed to bind to PBMNC at 4°C for 1 h to obtain surface labeling with minimal internalization. Aliquots of 106 cells were then placed into separate tubes and supernatants were separated from cells. Immediately after separation, cells underwent acid wash and acid-resistant radioactivity was determined by counting cell pellets after centrifugation. This procedure was repeated at various times after transferring cell aliquots to incubator at 37°C. Acid-resistant fraction was determined by dividing activity in cell pellet after acid wash by total activity on cell after 1 h of incubation at 4°C.

View larger version (30K): [in a new window]   FIGURE 4. Intracellular distribution of 125I-, 211At-, 111In-, 205,6Bi-, and 203Pb-labeled T101 in PBMNC. PBMNC incubated with radiolabeled T101 were suspended in TES buffer and disrupted. Cell nuclei and unbroken cells were removed by centrifugation at 250g. Supernatants were applied to Percoll/TES buffer and ultracentrifuged at 4°C for 60 min at 20,000g. Serial 0.5 mL fractions were collected from top and counted for radioactivity. Early fractions represent activity on cell surface and late fractions represent activity in lysosomes (fraction 14). Location of lysosome fraction was confirmed using enzyme marker ß-galactosidase (data not shown).