Antibody-drug conjugates (ADCs) are an increasingly popular modality for targeted drug delivery in many oncological and immunological applications. Recent advances in the field have optimized the chemical linker joining the drug and antibody, as many pharmacokinetic parameters including circulatory stability and payload release rate are dependent on this chemical linker. Recently, a leading peptide linker ValCitPABC has been demonstrated to be poorly stable in circulation, resulting in less efficacious ADCs due to premature payload release by endogenous proteases. Our group has developed an in vitro FRET-based screening assay which successfully identified a set of peptide linkers with promising stability profiles for ADC applications. Furthermore, our screening revealed a series of asparagine-containing linkers which we believe to be released by the lysosomal cysteine protease legumain, a novel vector for enzyme mediated drug release in ADCs. We have taken these results from our model system and evaluated ADCs that utilize these identified peptide linkers to assess the predictive nature of this assay towards ADC stability. Herein, we report a series of stability and efficacy studies of these novel linkers in tumor-targeting ADCs. Conjugating linker-fluorophore pairs to monoclonal antibody Trastuzumab enabled us to evaluate the lysosomal and serum stability of these Antibody-fluorophore conjugates (AFCs) by monitoring the loss of a fluorescent signal present on these conjugates over time. Compared to our FRET model system, we can establish a correlation between both data sets to conclude the accuracy of our assay on ADC stability. A synthetic route for the attachment of monomethyl auristatin E, a cytotoxic agent, was developed and performed on seven linkers to evaluate ADC efficacy. Conjugation of these linker payloads to Trastuzumab and Rituximab enabled the evaluation of targeted ADC cytotoxicity through IC50 determination on SK-BR-3 and Ramos cells using an XTT cell viability assay. The goal of these studies is to identify next-generation peptide linkers that have improved properties as compared to the current ValCitPABC linker.

Evaluation of novel peptide linkers for enhanced ADC stability and efficacy

ACS Fall 2020 Virtual Meeting

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