An estimated more than ninety-five percent of potential Alzheimer's Disease (AD) therapeutics and prophylactics have extremely limited ability to migrate from circulation to brain tissue. These transport limitations necessitate unfeasibly high doses of systemically administered drug to realize beneficial effects in the brain and/or promote off-target side effects throughout the body. A generalizable delivery technology that both targets transport-impaired drugs to and facilitates transport of these drugs across the blood brain barrier (BBB) would dramatically expand the armamentarium of agents for AD therapy and prophylaxis and could transform the way clinicians seek to treat and prevent AD by providing the breadth of pharmaceutical options needed to enable development of personalized AD treatment and prevention programs. `Trojan Horse' antibodies (Abs) that bind to proteins on and are transported across the BBB have been heavily pursued in central nervous system (CNS) drug targeting. Proteins bound by Trojan Horse Abs however, appear on both the BBB and other tissues throughout the body; less than one percent of systemically injected Trojan Horse Ab-drug conjugates reach the brain. We will realize step change improvements upon existing Trojan Horse Ab technologies by simultaneously identifying proteins and/or epitopes specific to or highly enriched on brain microvascular endothelial cells (BMECs) and generating human fibronectin domains (Fn3s), proteins that can possess Ab-like target binding affinity and specificity but are less time consuming and expensive than Abs to produce, that bind these BBB-specific molecular entities. Our innovative process for converting BMECs into water soluble nanometer-sized vesicles, known as CytoBits, uniquely positions us to succeed where others have encountered difficulties in engineering highly specific BBB-binding drug carrier proteins. Unlike whole cells, CytoBits are compatible with magnetic microsphere- and multicolor fluorescence activated cell sorting (FACS)-based methods that will enable us to screen a 250 million clone yeast-displayed Fn3 library and separate BMEC-binding clones from Fn3s that bind to cells derived from nontarget, e.g., lung and cardiac, tissues with fidelity that cannot be approached by the cell panning methods used in others' drug carrier protein development efforts. Leading candidate BBB-specific drug carrier Fn3s enriched via yeast-displayed Fn3 library screening will be expressed as soluble proteins and their affinities and specificities toward intact BMECs quantified via flow cytometry. Combining binding assay outcomes, measured rates of Fn3 endocytosis by BMECs, and utilization of tandem mass spectrometry to elucidate identities of BMEC proteins immunoprecipitated by drug carrier Fn3s will yield a dataset that will guide our choosing up to three BBB-specific Fn3s for mouse biodistribution studies carried out subsequent to this two-year project. Our long-term objective for this CNS drug targeting research initiative is to see BBB-specific drug carrier Fn3s broadly deployed in personalized AD drug programs by the year 2030; our team possesses both the animal study expertise and clinical connections needed to realize this timetable for translation. We are eager to continue our progress toward achieving this goal and are excited about the impact that its realization will have in making a difference for the millions of AD patients, friends, and family members whose lives have been tragically rearranged by this debilitating condition.
The extremely limited ability of an estimated more than ninety-five percent of potential Alzheimer's Disease (AD) therapeutics and prophylactics to migrate from the bloodstream to the central nervous system (CNS) necessitates unfeasibly high doses of systemically administered drug to realize beneficial effects within the brain and/or promotes off- target side effects throughout the body. Developing a generalizable delivery technology that both targets transport- impaired drugs to and facilitates transport of these drugs across the blood brain barrier would dramatically expand our inventory of effective AD pharmaceuticals and transform the way clinicians seek to treat and prevent AD by providing the breadth of options needed to enable development of personalized AD treatment and prevention programs through evaluation of patient responses to different drug combinations. In this work, we will utilize state-of-the-art protein engineering methods to develop brain-targeted AD drug carrier proteins that efficiently shuttle systemically administered AD drugs from circulation to the CNS and thus bring to bear the increased numbers of viable AD drug candidates that practitioners must have to harness the tremendous potential of personalized medicine in treating and preventing this incapacitating condition.
