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 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.
Highlights
We recently described a new ADC of the anti-CD30 mAb cAC10 [9], appended to the anti-tubulin agent, monomethyl auristatin E (MMAE), via a cathepsin-cleavable valine-citrulline linker [10, 11]
We have reported that cAC10 and cAC10-vcMMAE were comparable in binding to CD30ϩ cells and lacked interaction with CD30-negative cells [11]
The activities of the free drugs and the conjugates cAC10vc-MMAE and cAC10vc-monomethyl auristatin F (MMAF) were compared on CD30ϩ lymphoma cell lines including Karpas-299 and L540cy (Table 1)
Summary
We recently described a new ADC of the anti-CD30 mAb cAC10 [9], appended to the anti-tubulin agent, monomethyl auristatin E (MMAE), via a cathepsin-cleavable valine-citrulline (vc) linker [10, 11] This drug linker system was shown to be highly stable in vitro and in vivo [7, 12], and when applied to multiple mAbs, the resulting ADCs were selectively potent and effective against cognate antigen-positive tumor cells and tumor xenografts (LeY [8], CD30 [11], TMEFF2 [13], CD20 [14], and EphB2 [15]). These studies demonstrate the functional basis of drug delivery by ADCs with protease-cleavable linkers for anti-cancer therapeutics
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