Oxidizing potential of endosomes and lysosomes limits intracellular cleavage of disulfide-based antibody–drug conjugates

Abstract

Antibody-drug conjugate therapy entails targeted killing of cancer cells with cytotoxic compounds covalently linked to tumor-specific antibodies and shows promise in the treatment of several human cancers. Current antibody-drug conjugate designs that incorporate a disulfide linker between the antibody and cytotoxic drug are inspired by indirect evidence suggesting that the redox potential within the endosomal system is reducing. It is presumed that antigen-dependent endocytosis leads to disulfide linker reduction and intracellular release of free drug, but direct demonstration of such a mechanism is lacking. To determine whether the disulfide N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) linker would be reduced during endocytic recycling of the anti-HER2 antibody trastuzumab (Herceptin, Genentech), we synthesized a trastuzumab-SPP-Rhodamine red conjugate and developed a linker cleavage assay by using the self-quenching property of this fluorophore. In breast carcinoma SKBr3 cells, no SPP linker cleavage was observed, as detected by fluorescence dequenching upon internalization. By contrast, the conjugate did display fluorescence dequenching when diverted to the lysosomal pathway by geldanamycin, an effect partly due to proteolytic degradation rather than disulfide reduction. To understand why linker reduction was inefficient, we measured redox potentials of endocytic compartments by expressing a redox-sensitive variant of GFP fused to various endocytic proteins. Unexpectedly, we found that recycling endosomes, late endosomes, and lysosomes are not reducing, but oxidizing and comparable with conditions in the endoplasmic reticulum. These results suggest that intracellular reduction is unlikely to account for the potency of disulfide-linked antibodydrug conjugates.disulfide linker ͉ redox potential ͉ endocytosis ͉ HER2 ͉ Herceptin O ne approach to the treatment of cancer is to specifically target cytotoxic drugs to tumor cells by linking them via a cleavable linker to antibodies that recognize a tumor-restricted antigen. Such linkers include hydrazone linkers, designed to hydrolyze upon internalization into acidic endosomes and lysosomes (1-3); peptide linkers optimized for cleavage by certain lysosomal proteases (3-5); and disulfide linkers, thought to be cleaved by the reducing environment within the endocytic pathway (6-10). In the latter category, the monoclonal antibody C242 against CanAg (a glycotope on the mucin1 (MUC1) colorectal tumor antigen) has shown efficacy against colorectal xenograft models in vivo when disulfide-linked to the ribosomal inhibitor ricin A chain via a 4-succinimdyloxycarbonyl-methyl-␣-[2-pyridyldithio]-toluene (SMPT) linker (11) and to the maytansinoid-derived microtubule active drug DM1 via an N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) linker (12). The latter conjugate is now humanized and in clinical trials as cantuzumab mertansine (9, 13). An ideal linker should be cleaved only upon internalization of the antibody-drug conjugate into the tumor cell, thereby specifical...

Functional Requirements for the Lysosomal Thiol Reductase GILT in MHC Class II-Restricted Antigen Processing

“…However, reduction is not favored at the acidic pH found in the lysosomal compartment. In fact, lysosomes of adenocarcinoma lines were found to be oxidizing rather than reducing (29). The constitutive expression of GILT in APCs is likely to account for the enhanced reduction of proteins in the lysosomal compartment.…”

TRPM7 senses oxidative stress to release Zn 2+ from unique intracellular vesicles

“…6 B and C). This percentage is significantly greater than the percentage of oxidized roGFP in the cytosol or in mitochondria (∼11%) (55) but is much lower than the 96-97% observed in the ER lumen (57,58) or endolysosomes (94-97%) (57).…”

Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging

“…Finally, during preparation of this manuscript, a report was published (41) showing that a disulfide bond in the Trastuzumab-(S-S)-rhodamine red conjugate was not significantly reduced after endocytosis by breast carcinoma SKBr3 cells, suggesting a lack of redox function in the HER2-trafficking pathway. However, whether this inefficient processing of the disulfide could have derived from (i) different redox properties peculiar to the HER2 endosomal trafficking pathway or (ii) a lack of interaction between their macromolecular antibody conjugate and the endosomal redox enzymes responsible for disulfide reduction cannot be resolved from their data.…”