No abstract

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 valinecitrulline (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 antibodydrug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrinmediated 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.

Functional proteomics provides a powerful method for monitoring global molecular responses following activation of signal transduction pathways, reporting altered protein posttranslational modification and expression. Here we combine functional proteomics with selective activation and inhibition of MKK1/2, in order to identify cellular targets regulated by the MKK/ERK cascade. Twenty-five targets of this signaling pathway were identified, of which only five were previously characterized as MKK/ERK effectors. The remaining targets suggest novel roles for this signaling cascade in cellular processes of nuclear transport, nucleotide excision repair, nucleosome assembly, membrane trafficking, and cytoskeletal regulation. This study represents an application of functional proteomics toward identifying regulated targets of a discrete signal transduction pathway and demonstrates the utility of this discovery-based strategy in elucidating novel MAP kinase pathway effectors.