Exo-Cleavable Linkers: A Paradigm Shift for Enhanced Stability and Therapeutic Efficacy in Antibody-Drug Conjugates

Customized drug delivery systems have become paramount in the rapidly evolving field of precision medicine, and at the forefront of advances in this regard, antibody-drug conjugates (ADCs) present a symbiotic fusion of cytotoxic payloads and monoclonal antibodies (mAbs) facilitated by intricate chemical linkers. The search for ideal linkers that can dexterously provide the dual functionalities of enhancing circulatory stability and facilitating the effective release of the tumor payload is a present and formidable challenge. The valine-citrulline (Val-Cit) linker, which is used in a wide range of ADCs, despite its approval by the Food and Drug Administration, is associated with several inherent drawbacks, including hydrophobicity-induced aggregation, limited payload capacity, and premature payload release. This study presents a paradigm shift from the conventional linear linker archetype by introducing an exo-linker avant-garde approach that repositions the cleavable peptide linker at the exo-position of the PAB moiety. This molecular refinement not only offered the possibility to overcome the intrinsic drawbacks of the Val-Cit platform, but also significantly improved ADC stability, therapeutic efficacy, and pharmacokinetics. In vitro and in vivo biological evaluations, confirmed that ADCs designed using the exo-linker blueprint significantly attenuated premature payload release, while increasing the drug-to-antibody ratio, even with hydrophobic payloads, and this without inducing pronounced aggregation. Therefore, the fabricated exo-linker represents a significant improvement with respect to traditional Val-Cit ADCs. Moreover, under the influence of enzymes, such as carboxylesterases and human neutrophil elastase, the payload remained stably conjugated to the ADC, underscoring a favorable safety profile and highlighting potential for clinical translatability. Thus, our findings also demonstrate the potential of the novel exo-linker paradigm as well as the profound implications of nuanced molecular modifications for reshaping ADC design and functionality.