Cathepsin B Processing Is Required for the <i>In Vivo</i> Efficacy of Albumin–Drug Conjugates

Abstract

Targeted drug delivery approaches that selectively and preferentially deliver therapeutic agents to specific tissues are of great interest for safer and more effective pharmaceutical treatments. We investigate whether cathepsin B cleavage of a valine-citrulline [VC(S)] containing linker is required for the release of monomethyl auristatin E (MMAE) from albumin-drug conjugates. In this study, we use an engineered version of human serum albumin, Veltis® High Binder II (HBII), which has enhanced binding to the neonatal Fc receptor (FcRn) to improve drug-release upon binding and FcRn-mediated recycling. The linker–payload was conjugated to cysteine 34 of albumin using a carbonylacrylic (caa) reagent which produced homogenous and plasma stable conjugates that retained FcRn binding. Two caa–linker–MMAE reagents were synthesised — one with a cleavable [VC(S)] linker and one with a non-cleavable [VC(R)] linker — to question whether protease-mediated cleavage is needed for MMAE release. Our findings demonstrate that cathepsin B is required to achieve efficient and selective anti-tumour activity. The conjugates equipped with the cleavable [VC(S)] linker had potent anti-tumour activity in vivo facilitated by the release of free MMAE upon FcRn binding and internalisation. In addition to the pronounced anti-tumour activity of the albumin conjugates in vivo, we also demonstrated their preferable tumour biodistribution and biocompatibility with no associated toxicity or side effects. These results suggest that the use of engineered albumins with high FcRn binding combined with protease cleavable linkers is an efficient strategy to target delivery of drugs to solid tumours.