The
transferrin cycle was used to attempt the import of bioactive macromolecules into cells with the aid of an
acid-labile cross-linking agent. Anti-
tetanus F(ab')2 fragments were iodinated and then conjugated to
transferrin with a newly developed
acid-labile cleavable
cross-linking reagent,
bismaleimidoethoxy propane, following thiolation of both
proteins. Noncleavable conjugates were also prepared. At saturating conjugate concentrations, the uptake rate for both conjugates averaged over the first 2 h is about 6.5 fmol/million cells/min. Incubation of loaded cells in fresh medium for 30 min and analysis of cell pellets and supernatants reveal that 1) of the previously cell-associated label, only intact conjugate (about 50% of the label) is returned to the medium; 2) most of the remaining cell-associated material for the cleavable conjugate is chromatographically coincident with free Fab with some contribution from free F(ab')2 fragments. In contrast, the cell pellets loaded with noncleavable conjugates contained intact
transferrin-F(ab'), conjugates. These results are consistent with
transferrin receptor-mediated uptake of
acid-labile conjugate followed by hydrolysis in acidified endosomes and resulting in concentration of free F(ab')2 and Fab within a prelysosomal intracellular compartment. A
protein shuttle such as
transferrin may therefore be used with ketal based
acid-labile cross-linkers to load foreign molecules into an intracellular compartment. In addition, these data provide independent confirmation of the low pH compartment within the
transferrin cycle. This new methodology is applicable to other cases of receptor/
ligand trafficking to report low pH compartments independent of morphological analysis. Since
transferrin receptors are overexpressed in
tumors,
antineoplastic agents could be targeted to
tumors as
transferrin acid-labile conjugates. This import system might be particularly useful in combatting the
tumor cell export of
antitumor agents occurring in multidrug resistance.