The chimeric anti-CD30
monoclonal antibody cAC10, linked to the
antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-
tumor activity in vitro and in vivo. These drugs are appended via a
valine-
citrulline (vc)
dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal
cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two
antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30
surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and
antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of
clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-
antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified
cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug.
Cysteine protease inhibitors, but not aspartic or
serine protease inhibitors, blocked
antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes,
cathepsin B and perhaps other lysosomal
cysteine proteases are requisite for drug release and provide a mechanistic basis for developing
antibody-drug conjugates cleavable by intracellular
proteases for the targeted delivery of anti-
cancer therapeutics.