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Antibody-drug conjugates (ADCs) are a class of drugs used for targeted delivery in the treatment of cancer. The prototypical linker used for such ADCs is the lysosomally cleaved Val-Cit-PABC linker: emerging as a result of its rapid cleavage rate by the lysosomal enzyme cathepsin B as well as its stability in human plasma. However, recent studies have shown that this system is frequently unstable in the presence of various enzymes including neutrophil elastases and carboxylesterases. To mitigate this issue, we have designed a peptide library that can be readily screened in order to identify sequences with improved properties. In short, the library was designed to utilize a turn-on fluorescence assay: a simple assay made possible by a fluorophore, AMC (7-Amino-4-methylcoumarin), known to be non-fluorescent when bound to a peptide, but highly fluorescent upon cleavage. 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 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.



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Design and optimization of a turn-on fluorescence assay for the identification of improved ADC linkers