The ValCit dipeptide has been the standard linker chemistry utilized in antibody drug conjugate technology, but has recently revealed instabilities resulting in undesirable, premature payload release. Previously, our group screened an array of dipeptide chemical linkers in lysosomal and serum environments leading to the identification of a series of asparagine containing linkers with desirable biorthogonal profiles. We an aspaginyl endoprotease (Legumain) as responsible for the lysosomal cleavage of these linkers. Our study of legumain-cleavable linkers led us to propose the direct release of a payload from Asn-containing linkers, as opposed to the release of a PABC spacer and subsequently self-immolation. This strategy would decrease the synthetic steps required for linker payload synthesis and would increase the hydrophilicity of the linker payload, contributing reduced ADC aggregation. <br/>Herein, we report the synthesis and utility of MMAE ADCs utilizing a variety of asparagine-based linkers. A mono and dipeptide asparagine linker payloads were evaluated against the non-cleavable mc linker to validate legumain activity to release the desirable payload. Lysosomal catabolism and serum stability evaluated the biorthogonality of these novel asparagine linkers and validated the release of MMAE from the system. Lastly, Cytotoxicity of these linker-payloads was determined to demonstrate the efficacy of ADCs in a biological setting. Together, these impressive profiles of Legumain cleavable ADCs give credence to their utility and next-generation capacity for the future of ADC linker development.

Expanding the utility of legumain cleavable ADC linkers through direct payload coupling

ACS Spring Meeting 2021

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