Amyloidosis is a devastating pathology that is associated not only with the development of Alzheimer's disease, but also with the lesser known, but similarly devastating, disorder immunoglobulin light chain- associated (AL) amyloidosis. Patients with AL develop amyloid in abdominothoracic organs and peripheral nerves leading to organ dysfunction which is often fatal. The amyloid deposits in systemic diseases are immunologically inert – they are not recognized or cleared by phagocytic cells of the immune system (macrophages [Mφ]) and do not illicit an antibody response. In patients presenting with significant cardiac amyloidosis, the prognosis is poor with a median survival of ~9 mos. Treatment of AL amyloidosis generally involves anti-plasma cell chemotherapy and immunotherapy to suppress plasma cell secretion of the amyloid-forming light chain protein. However, clearance of tissue amyloid has now become a major goal of many of the novel therapeutics being developed for these patients. Consequently, amyloid-reactive monoclonal antibodies have been developed that can be used to opsonize deposits and induce clearance by Mφ. Despite disappointing results from late stage clinical evaluation of certain of these mAbs, these trials demonstrated that clearance of amyloid and improvement in organ function can be achieved. The goal of this exploratory proposal is to generate two chimeric antigen receptor (CAR) constructs, expressed in macrophages, to induce specific recognition and enhanced phagocytosis of AL amyloid. To facilitate amyloid binding, the CARs will incorporate either the scFv domain of the amyloid reactive 11-1F4 mAb or the multi-amyloid binding peptide p5+14. The cytoplasmic domain of the CAR, from the murine FcR, will be incorporated to signal phagocytosis of the amyloid following receptor binding. Phagocytosis of synthetic AL amyloid fibrils and patient-derived AL amyloid extracts (labeled with the pH-sensitive dye pHrodo red) by CAR-phagocytic (P) Mφ will be quantified in vitro by monitoring the increased fluorescence emission of the fluorophore as the labeled amyloid enters the acidified phagolysosome. Additionally we will use small animal optical imaging to study co-localization with, and phagocytosis of, fluorophore-labeled human amyloid by CAR- P Mφ in mice. Quantitatively significant improvement in phagocytosis of AL amyloid due to the presence of the CAR will be assessed by comparison with CAR-negative Mφ and direct opsonization of amyloid using 11-1F4. The amyloid-directed CAR-P Mφ is a novel approach to the treatment of AL and related systemic amyloidoses. The long term goal of this study is to provide support for an engineered, cell-based amyloid clearance paradigm for the treatment of amyloidosis. In light of positive data from these studies, we hope that the amyloid-directed CAR-P technology can be optimized for efficacy and utility, and translated to the clinic to provide meaningful benefit to patients by enhancing quality ...