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Antibody-drug conjugates (ADCs) are a class of drug currently used for the targeted treatment of cancer. The prototypical linker used for such ADCs is the Val-Cit-PABC linker due to its rapid cleavage rate by the lysosomal enzyme cathepsin B as well as its stability in human plasma. However, recent studies have shown this system to be unstable in the presence of various enzymes such as carboxylesterases and neutrophil elastases. To mitigate this issue, we designed a peptide library that can be readily screened in order to identify linker sequences that are still rapidly cleaved by lysosomal enzymes but are stable in human and mouse plasma. In short, the library was designed to utilize a turn-on fluorescence assay made possible by the fluorophore AMC (7-Amino-4-methylcoumarin). AMC is known to be non-fluorescent when the 7-amino group is bound as an amide but is highly fluorescent upon cleavage of the amide bond. Therefore, AMC can be employed as a fluorescent probe for rapid determination of amide bond cleavage – specifically that of ADC linkers. Lysosomal ADC processing relies on cleavage of the amide bond between the linker and the cytotoxic payload, and therefore the turn-on fluorescence assay provides a simple method for determining whether or not particular peptide linkers are susceptible to such cleavage. Due to its poor nucleophilicity, the AMC was attached to a single amino acid and subsequently coupled to variable tripeptide linkers. All individual compounds were purified and characterized by LCMS resulting in 130 linkers for screenings. We report the results of the linker stability and plasma stability studies focusing on linkers that have the best potential for incorporation in ADC designs.



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Design of a Turn-on Fluorescence Assay for the Identification and Application of Improved ADC Linkers