Development of chimeric antigen receptor-expressing macrophages for enhanced phagocytosis of systemic amyloid

淀粉样变性是一种破坏性的病理，不仅与阿尔茨海默氏症的发展有关， 疾病，但也与鲜为人知的，但同样具有破坏性的，疾病免疫球蛋白轻链- 相关性淀粉样变性。AL患者在胸腹器官和外周中发生淀粉样蛋白 导致器官功能障碍，这往往是致命的。全身性疾病中的淀粉样蛋白沉积是 免疫惰性-它们不被免疫系统的吞噬细胞识别或清除 （巨噬细胞[Mφ]），并且不引起抗体应答。在患有严重心脏病的患者中， 淀粉样变性，预后差，中位生存期约9个月。 AL淀粉样变性的治疗通常涉及抗浆细胞化疗和免疫治疗， 抑制浆细胞分泌淀粉样蛋白形成轻链蛋白。然而，组织的清除 淀粉样蛋白现在已经成为为这些患者开发的许多新疗法的主要目标。 因此，淀粉样蛋白反应性单克隆抗体已被开发，可用于调理 沉积并诱导Mφ清除。尽管晚期临床评价的结果令人失望， 这些试验表明，某些这些单克隆抗体，清除淀粉样蛋白和改善器官功能， 可以实现。 该探索性提议的目标是产生两个嵌合抗原受体（CAR）构建体， 在巨噬细胞中表达，以诱导AL淀粉样蛋白的特异性识别和增强的吞噬作用。到 当CAR促进淀粉样蛋白结合时，汽车将掺入淀粉样蛋白反应性11- 1F 4的scFv结构域， mAb或多淀粉样蛋白结合肽p5+14。来自鼠FcR的CAR的胞质结构域， 将被掺入以在受体结合后发出淀粉样蛋白吞噬的信号。吞噬合成 AL淀粉样蛋白原纤维和患者来源的AL淀粉样蛋白提取物（用pH敏感染料pHrodo red标记）， CAR-吞噬细胞（P）Mφ将通过监测CAR-吞噬细胞（P）Mφ的增加的荧光发射在体外定量。 荧光团作为标记的淀粉样蛋白进入酸化的吞噬溶酶体。此外，我们将使用小动物 光学成像，以研究与荧光团标记的人淀粉样蛋白的共定位和CAR- 小鼠P Mφ。由于存在的蛋白质，AL淀粉样蛋白的吞噬作用在数量上显著改善。 将通过与CAR阴性Mφ和使用11- 1F 4的淀粉样蛋白的直接调理作用进行比较来评估CAR。 淀粉样蛋白导向的CAR-PM φ是治疗AL和相关系统性疾病的新方法。 淀粉样变性这项研究的长期目标是为工程化的、基于细胞的淀粉样蛋白提供支持 清除范例用于治疗淀粉样变性。根据这些研究的积极数据，我们希望， 淀粉样蛋白导向的CAR-P技术可以优化疗效和实用性，并转化为临床， 通过提高生活质量和延长患者生存期为患者提供有意义的获益。

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 of life and prolonging patient survival.