Antibody drug-conjugate (ADC) technology is a rapidly evolving modality for targeted drug delivery. Much of the success of ADCs is due to technical advances made in the design of the peptide linkage between the antibody and the therapeutic payload. This linkage has been optimized to be stable in circulation while maintaining a rapid release mechanism that is specific to lysosomal conditions. However, some ADCs have exhibited stability problems due to high solvent exposure of the linker-payload to the surrounding environment which can result in premature cleavage of the linkage by endogenous proteases in circulation, thus leading to off-target toxicity. For example, the prototypical ADC linker ValCitPABC has been shown to undergo premature cleavage by esterases and by neutrophil elastases, thus driving some adverse effects. The goal of this project is to develop a new generation of proteolytically cleavable linkers that offer increased stability to extracellular esterases and proteases.<br/><br/>This investigation into the stability of various peptides linkages utilizes a high-throughput assay involving a fluorophore and quencher pair attached to opposing ends of a tripeptide linker, thus simulating the payload and antibody respectively. Through Förster resonance energy transfer (FRET), we can observe cleavage through the evolution of a fluorescein signal over time. Seventy-five peptide-linked FRET pairs were synthesized and purified through preparative HPLC. Herein, we will describe the results of these studies and will report several peptide sequences that appear to offer improved properties as compared to the traditional ValCitPABC linker. Lability of FRET pairs was tested against lysosomal protease Cathepsin B and tritosomes to ensure that the linkage is cleaved, thus predicting payload release after receptor-mediated endocytosis. Stability of linker against Ces1C and neutrophil elastase confirmed the linkage is stable before internalization and thus little premature payload release is expected. Stability was further evaluated in mouse plasma and human plasma to mirror physiological circulatory conditions to ensure cleavage was not observed. Ultimately, this work will help to identify novel peptide linkages that are stable under physiological conditions but are rapidly cleaved upon lysosomal uptake.

Optimization of ADC linkers: Design and evaluation of a FRET-based ADC linker-library

Macromolecular Chemistry: The Second Century

[Author Benefits](https://scimeetings.acs.org/?utm_source=pubs_content_marketing&utm_medium=website&utm_campaign=0320_MCF_NPI_Launch_Spring_Homepage&ref=pubs_content_marketing)

[How to use SciMeetings](https://storage.googleapis.com/dl.morressier.com/acs/SciMeetings-ACSSpring2020-HowToGuide_M3020-Final.pdf)

[Frequently Asked Questions](https://storage.googleapis.com/dl.morressier.com/acs/SciMtng_ACSNMSp2020_FAQ_M3120_Final.pdf)

ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.