PROJECT SUMMARY/ABSTRACT The precise pathophysiology of Alzheimer’s disease (AD) remains unknown, but the development of amyloid- beta (Aβ) plaques is thought to be a key initiating event preceding the formation of neurofibrillary tangles and clinical disease. Anti-Aβ monoclonal antibodies (mAb) are the first disease modifying therapies approved for AD, but clinical trials showed significant and potentially fatal dose-limiting side effects, including amyloid-related imaging abnormalities (ARIA) that can manifest as edema and microhemorrhage. Peripheral monocytes/macrophages enter the brain and associate with Aβ plaques in the APP/PS1 transgenic mouse model of AD. These cells can reduce plaque load in vivo despite making up a small percentage of plaque-associated macrophages, suggesting that a better mechanistic understanding of the recruitment and function of these cells in the AD brain may improve anti-Aβ therapies. In preliminary work, a chimeric antigen receptor (CAR) was expressed in macrophages (CAR-Ms) containing an extracellular Aβ binding domain and an intracellular Fc receptor signaling domain to enhance the ability of these cells to phagocytose Aβ. Aβ CAR-Ms reduced plaque load ex vivo on amyloid-laden APP/PS1 brain slices, and in vivo when injected into the hippocampus of APP/PS1 mice; however, plaque reduction was confined to the region immediately surrounding the cell injection site and CAR-M migration was minimal, limiting its efficacy. The objective of this proposal is to enhance the efficacy of Aβ CAR-Ms by identifying factors that promote the infiltration of peripheral monocytes into the brain and their migration within the brain parenchyma. This proposal will test the hypothesis that improving brain infiltration and migration of Aβ CAR-Ms will improve Aβ clearance in aged APP/PS1 mice without increasing ARIA. An important theoretical advantage of CAR-M therapy compared to mAb therapy is the active degradation of plaque material, potentially mitigating concerns for ARIA. Aim 1 will determine whether CX3CR1 overexpression allows CAR-Ms to better infiltrate the brain from the periphery and characterize brain-penetrating CAR-Ms. This aim will further investigate the effect of Aβ CAR-Ms on the composition and phenotype of the brain cellular microenvironment. Aim 2 will compare the safety and efficacy of CAR-M therapy to mAb therapy in APP/PS1 mice. While mouse models do not replicate the entire spectrum of human ARIA, the frequency of microhemorrhages after CAR-M or mAb treatment will be quantified as a measure of ARIA-like phenomena. These studies will further develop and characterize a novel form of AD therapy and provide insights in basic macrophage biology. I will pursue these studies under the guidance of a team of physician-scientists, including a sponsor with expertise in CAR and macrophage biology, and a co-sponsor with over two decades of experience in AD mouse modeling. My training plan incorporates inter-departmental exper...