Tumor Delivery and In Vivo Processing of Disulfide-Linked and Thioether-Linked Antibody−Maytansinoid Conjugates

Abstract

Antibody-drug conjugates (ADCs) are designed to eradicate cancer cells that express the target antigen on their cell surface. A key component of an ADC is the linker that covalently connects the cytotoxic agent to the antibody. Several antibody-maytansinoid conjugates prepared with disulfide-based linkers such as those targeting the CanAg antigen have been shown to display more activity in preclinical mouse xenograft models than corresponding conjugates prepared with uncleavable thioether-based linkers. To investigate how the linker influences delivery and activation of antibody-maytansinoid conjugates, we isolated and characterized the [(3)H]maytansinoids from CanAg-positive tumor tissues following a single intravenous administration of 300 microg/kg (based on maytansinoid dose) of anti-CanAg antibody (huC242)-(3)H-maytansinoid conjugates prepared with cleavable disulfide linkers and an uncleavable thioether linker. We identified three target-dependent tumor metabolites of the disulfide-linked huC242-SPDB-DM4, namely, lysine-N(epsilon)-SPDB-DM4, DM4, and S-methyl-DM4. We found similar metabolites for the less hindered disulfide-linked huC242-SPP-DM1 conjugate with the exception that no S-methyl-DM1 was detected. The sole metabolite of the uncleavable thioether-linked huC242-SMCC-DM1 was lysine-N(epsilon)-SMCC-DM1. The AUC for the metabolites of huC242-SMCC-DM1 at the tumor over 7 d was about 2-fold greater than the corresponding AUC for the metabolites of the disulfide-linked conjugates. The lipophilic metabolites of the disulfide-linked conjugates were found to be nearly 1000 times more cytotoxic than the more hydrophilic lysine-N(epsilon)-linker-maytansinoids in cell-based viability assays when added extracellularly. The cell killing properties associated with the lipophilic metabolites of the disulfide-linked conjugates (DM4 and S-methyl-DM4, and DM1) provide an explanation for the superior in vivo efficacy that is often observed with antibody-maytansinoid conjugates prepared with disulfide-based linkers in xenograft mouse models.

A New Triglycyl Peptide Linker for Antibody–Drug Conjugates (ADCs) with Improved Targeted Killing of Cancer Cells

“…Furthermore, in several EGFR-expressing xenograft models and in an EpCAM-expressing xenograft model, the CX ADC was significantly more active in vivo than the SMCC ADC. The SMCC ADC requires lysosomal proteolysis of the antibody scaffold to release a polar catabolite, lysine-SMCC-DM1, which bears both a negative and a positive charge (11,14,28). In contrast, the lysosomal processing of the CX ADC requires cleavage at only one of the amide bonds in the triglycyl linker to generate maytansinoid catabolites bearing carboxylic acid group (DM-CX1, DM-CX2).…”

“…The noncleavable linker-bearing ADCs undergo proteolysis of their antibody component in lysosomes to release cytotoxic lysine-or cysteine-linked payload catabolites (11)(12)(13). Upon ultimate efflux out of target cells, these polar catabolite molecules, which bear both a positive and a negative charge, cannot easily enter and kill neighboring cells and therefore lack bystander-killing activity (11,14,15).…”

“…The sterically hindered disulfide bond in the DM4 conjugate with SPDB or charged sulfo-SPDB linker is stable to cleavage during circulation, while allowing disulfide reduction and S-methylation of the polar lysine-linked DM4 catabolite inside target cells to generate uncharged DM4 and S-methyl DM4 molecules (11,16,17). Upon efflux from target cells, the uncharged maytansinoid molecules can enter and kill neighboring bystander cells that do not express target antigen (11,14,15). The VC-MMAE peptide linkage (used in brentuximab vedotin) is cleaved in lysosomes and upon further PABC spacer-immolation releases cytotoxic amine-bearing MMAE, which is cell-permeable and can exert bystander cytotoxic activity toward neighboring cells (12,18).…”

The Effect of Different Linkers on Target Cell Catabolism and Pharmacokinetics/Pharmacodynamics of Trastuzumab Maytansinoid Conjugates

“…The ULTRA-TURRAX T8 dispersing instrument with an S8N dispersing tool was obtained from IKA Works Inc. Trastuzumab and the humanized antiglycoprotein D (gD) control antibody, 5B6, were prepared at Genentech. Antibody-[ 3 H]maytansinoid conjugates were prepared at ImmunoGen, Inc., as described previously (12). The ratio of linked …”

“…A stable tritium label was incorporated into the C-20 methoxy group of the DM1 used to prepare the 3 H-labeled conjugates as described previously (12). HER2-dependent processing of the 3 H-labeled conjugates was investigated in HER2-overexpressing BT-474EEI, MCF7-neo/HER2, and SK-BR-3.…”