LC-MS/MS Quantification and Solvent-Exposure Modeling Reveal a Mismatch Between Linker Exposure and Payload Release in Wild-Type Trastuzumab-VC-PABC-MMAE in Rat Plasma.
TL;DR
A mismatch between modeled exposure and observed release argues against a simple exposure-driven cleavage mechanism as the primary explanation for the wild-type behavior and suggests that the observed payload release is influenced by bond chemistry in addition to linker exposure.
Abstract
Free monomethyl auristatin E (MMAE) released from trastuzumab-valine-citrulline-p-aminobenzyl carbamate (VC-PABC)-MMAE conjugates was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after incubation in rat plasma at 37°C for 4 days. A wild-type conjugate prepared by maleimide-thiol interchain cysteine conjugation showed 4% ± 3.5% release (n = 3 for each), whereas a Lys188-selective comparator prepared by click chemistry after lipoic acid ligase A-mediated modification showed 4% ± 3.5% release. To examine whether this difference could be explained by linker-payload exposure, molecular dynamics simulations were performed, and solvent-accessible surface areas (SASA) were calculated for representative wild-type interchain cysteine environments and for the Lys188 conjugate. Mean SASA values for the wild-type models were 1132-1293 Å2, all lower than that of the Lys188 model (1524 Å2). Thus, the more stable comparator exhibited the highest modeled exposure. This mismatch between modeled exposure and observed release argues against a simple exposure-driven cleavage mechanism as the primary explanation for the wild-type behavior. Instead, the results suggest that the observed payload release is influenced by bond chemistry in addition to linker exposure.