PROJECT SUMMARY The blood-brain barrier (BBB) poses the greatest challenge for developing effective therapies for neurological diseases. Inspired by receptor-mediated transcytosis, bi-specific antibodies (bsAbs) against transferrin receptor (TfR) have demonstrated significant improvements of CNS delivery. However, the overall brain penetration was still modest, with large majority of administrated bsAbs remain in blood. As transcytosis at BBB is a bi-directional process and inevitably leads anti-TfR bsAb reaching a concentration equilibrium between the blood and the brain sides, we hypothesize that by minimizing abluminal-to-luminal efflux, the concentration equilibrium can be shifted toward BBB penetration. Our design principle is to fuse the variable fragment (Fv) of anti-TfR to the N-terminal of a therapeutic IgG, via cleavable linker(s) specific to disease-associated protease present in the brain. Once delivered to the brain by TfR-mediated transcytosis, therapeutic IgG will be activated and stays at the brain side because it loses binding ability to TfR. Released anti-TfR Fv will transport back to the blood side then be eliminated by renal clearance. TfR-bound prodrugs will be further transcytosed and thus forming a net flow of therapeutic Ab penetration from blood to brain. Our long-term goal is to develop a highly efficient BBB delivery approach that enables effective treatments of neurological disorders such as brain cancer and neurodegenerative diseases. The objective of this MPI R21 project is to prove the concept of this novel BBB delivery technology based on protease-activated prodrug designs. We will use cathepsin S activated anti-amyloid β (Aβ) for Alzheimer’s disease (AD) as the model system in this study. Building on our collective expertise on protein engineering, protease biochemistry, BBB transportation and AD, we will, Aim 1: design, construct and optimize protease-activated bi-specific antibody prodrugs; and Aim 2: validate BBB penetration and therapeutic efficacy of antibody prodrugs using mouse models of cerebral amyloid angiopathy (CAA). The approaches are innovative, because the protease-cleavable prodrug designs can prevent the reverse transcytosis, shift the concentration equilibrium, and thus promote therapeutics penetration from blood to brain. The proposed research is significant because it develops a platform technology enabling to (1) improve BBB penetration of biologics including monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), (2) greatly reduce off-site on- target side effects by in situ activation in brain, and (3) treat a variety of neurological disorders currently non- targetable.