Project Summary Sporadic, late onset Alzheimer disease (LOAD) is the most common form of aging-dependent neurodegenerative disorder. LOAD is associated with deposition of extracellular amyloid plaques and intra-neuronal neurofibrillary tangles (NFT), formed by Aβ peptides and phosphor- tau, respectively, in the central nervous system (CNS). Although there is strong consensus that toxic forms of Aβ and tau cause cognitive impairments, efforts to halt or reverse cognitive decline in LOAD patients targeting Aβ, plaques and tangles have been ineffective and emphasize an unmet need for therapeutic approaches that are not based on LOAD-associated anatomical pathologies. Several data implicate neuroinflammation, microglia dysfunction and supraphysiological TNFα activity in LOAD pathogenesis. These include new evidence generated by Dr. D’Adamio, the cofounder of NanoNewron LLC, indicating that supraphysiological TNF-α increases excitatory/inhibitory balance and impairs Long-Term Potentiation (LTP), a form of synaptic plasticity that represents a cellular model for learning and memory. These changes occur early and are independent of changes in Aβ levels and brain pathology. Importantly, resetting TNFα activity at physiological levels using low doses of anti-rat-TNF-α neutralizing antibody normalizes these alterations. The evidence that peri-spinal and intrathecal administration of FDA-approved biologic TNFα– specific inhibitors (TNFI) improved cognition in LOAD patients further emphasizes the therapeutic potential of targeting supraphysiological TNFα in LOAD. Unfortunately, FDA- approved biologic TNFIs have limited blood-brain barrier (BBB) penetration because of the large size. To overcome this BBB-penetrance obstacle, NanoNewron LLC proposes a Phase I STTR project to characterize anti-TNF-α camelid nanobodies that neutralize TNF-α activity (TNFI- Nabs), with the goal of preventing, arresting or reversing LOAD. Given the small size and structure, nanobodies can penetrate the CNS much more rapidly than conventional antibodies. Moreover, BBB permeability can be increased by targeting the nanobody to the transferrin receptors on the surface of microvascular endothelial cells, favoring transcytosis, and/or by increasing the isoelectric point. Thus, NanoNewron has also developed monovalent TfR-Nabs that will be used to shuttle TNFI-Nabs efficiently into the CNS. The results of this study will serve as a stepping-stone for NanoNewron’s efforts to develop TNFI-Nabs disease modifying therapeutic candidates for LOAD.

Project Narrative Neuroinflammation, microglia dysfunction and supraphysiological TNFα activity have been implicated in sporadic late onset Alzheimer Disease (LOAD) pathogenesis but, unfortunately, FDA-approved inhibitors of TNFα are large molecules that have limited blood-brain barrier penetration. In this application, we propose to identify single domain antibodies with high blood- brain barrier penetrability for blocking supraphysiological TNFα activity in the Central Nervous System (TNFI-Nab) as therapeutic agents to prevent, arrest or reverse LOAD. Given that there is no cure for SAD, that ~5.4 million Americans have LOAD and that without disease-modifying therapeutics this number is expected to triple in 2050, this study addresses a critical medical area with a pressing need for therapeutic advances.