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Updated: Aug 6, 2026
Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction
Published on: September 14, 2018
LC-MS/MS Quantification and Solvent-Exposure Modeling Reveal a Mismatch Between Linker Exposure and Payload Release
Kazutoshi Takahashi1, Shunsuke Yamazaki1, Yutaka Matsuda1
1Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan.
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.
Insights
Free monomethyl auristatin E (MMAE) release from antibody-drug conjugates was studied. Payload release was similar between wild-type and Lys188-selective conjugates, despite differing linker exposures, suggesting bond chemistry influences drug release.
Area of Science:
- Bioconjugation Chemistry
- Pharmacology
- Computational Chemistry
Background:
- Antibody-drug conjugates (ADCs) are crucial therapeutics.
- Understanding payload release mechanisms is vital for ADC efficacy and safety.
- Linker stability and payload exposure influence ADC pharmacokinetics.
Purpose of the Study:
- To quantify free monomethyl auristatin E (MMAE) release from trastuzumab-based ADCs.
- To investigate the role of linker exposure in payload release.
- To compare release mechanisms between wild-type and Lys188-selective conjugates.
Main Methods:
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for MMAE quantification.
- Incubation in rat plasma at 37°C for 4 days.
- Molecular dynamics simulations and solvent-accessible surface area (SASA) calculations.
Main Results:
- Similar MMAE release (4% ± 3.5%) observed for both wild-type and Lys188-selective conjugates.
- Lys188-selective conjugate showed higher modeled solvent-accessible surface area (1524 Ų) compared to wild-type (1132-1293 Ų).
- Mismatch between higher modeled exposure and similar observed release in the Lys188 conjugate.
Conclusions:
- Payload release is not solely driven by linker exposure.
- Bond chemistry plays a significant role in MMAE release from ADCs.
- Further investigation into linker-payload interactions is warranted.
