Cathepsin B Processing Is Required for the In Vivo Efficacy of Albumin–Drug Conjugates

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 investigated 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 used an engineered version of human serum albumin, Veltis High Binder II (HBII), which has enhanced binding to the neonatal Fc (fragment crystallizable) 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 homogeneous and plasma stable conjugates that retained FcRn binding. Two caa–linker–MMAE reagents were synthesized─one with a cleavable [VC(S)] linker and one with a noncleavable [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 antitumor activity. The conjugates equipped with the cleavable [VC(S)] linker had potent antitumor activity in vivo facilitated by the release of free MMAE upon FcRn binding and internalization. In addition to the pronounced antitumor activity of the albumin conjugates in vivo, we also demonstrated their preferable tumor 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 tumors.