Antibody-Drug Conjugates (ADC) have become an important therapeutic approach for delivering highly cytotoxic drugs to tumor while sparing normal tissue. The field has made great progress with two ADCs approved by FDA and over 20 other examples in clinical trials. In spite of these advances, improving ADC therapeutic window remains a major challenge. A key component of ADC design is the linker connecting the payload and the antibody. Peptide-based linkers (eg. Valine-Citrulline) that are cleavable by lysosomal proteases have shown sufficient serum stability and rapid payload-release in targeted cells. It’s conceivable that if the linker can be tailored more toward tumor-specific proteases, a better therapeutic index may be obtained. However, peptide-based linker scaffolds afford only limited space for modulating protease cleavability. We now report the discovery of a set of non-peptide linkers with restricted protease cleavability. Using computational modeling, novel peptidomimetic linkers were designed as preferential cathepsin B substrates. SAR of cleavage kinetics as well as crystallographic structure analysis subsequently confirmed the design hypothesis. Using protease inhibitors, we found that cleavage of these peptidomimetic linkers was indeed more restricted to cathepsin-B like proteases compared to the peptide linkers. We compared ADCs containing the non-peptide and peptide linkers in terms of in vitro activity, payload release, in vivo stability and efficacy using auristatin MMAE and DNA-damaging payloads. Despite their restricted protease cleavability, we found these non-peptide linkers can serve as effective ADC components . The implications of our findings regarding in vivo release kinetics for protease-cleavable linkers will also be discussed.

Computational design of non-peptide linkers with restricted protease cleavability for antibody-drug conjugates

251st National Meeting (2016)

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