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

淀粉样变性是一种毁灭性的病理，不仅与阿尔茨海默氏症的发展有关 疾病，但也与鲜为人知，但同样具有破坏性的免疫球蛋白轻链紊乱- 相关性(AL)淀粉样变性。AL患者的腹胸器官和外周出现淀粉样蛋白 神经导致器官功能障碍，这通常是致命的。系统性疾病中的淀粉样沉积是 免疫惰性-免疫系统的吞噬细胞不能识别或清除它们 (巨噬细胞[Mφ])，不违法抗体反应。在表现为显著心脏疾病的患者中 淀粉样变性预后较差，中位生存期约9个月。 AL淀粉样变性的治疗一般包括抗浆细胞化疗和免疫治疗 抑制浆细胞分泌形成淀粉样蛋白的轻链蛋白。然而，组织的清除 淀粉样蛋白现在已经成为许多正在为这些患者开发的新疗法的主要目标。 因此，已开发出可用于调理的淀粉样蛋白反应性单抗。 沉淀物和诱导Mφ清除。尽管晚期临床评估结果令人失望，但 其中某些单抗，这些试验表明淀粉样蛋白的清除和器官功能的改善 是可以实现的。 这一探索性方案的目标是产生两个嵌合抗原受体(CAR)结构， 在巨噬细胞中表达，诱导对AL淀粉样蛋白的特异性识别和增强吞噬作用。至 促进淀粉样蛋白结合，CARS将整合淀粉样蛋白反应性11-1F4的scFv结构域 MAb或多淀粉样结合肽P5+14。CAR的细胞质结构域，来自小鼠FCR， 将在受体结合后加入以发出吞噬淀粉样蛋白的信号。人工合成的吞噬功能 Al淀粉样纤维和患者来源的AL淀粉样蛋白提取物(用pH敏感染料pHrodo Red标记) 体外将通过监测巨噬细胞(P)Mφ增强的荧光发射来对其进行定量 荧光团作为标记的淀粉样蛋白进入酸化的吞噬小体。此外，我们还将使用小动物 用CAR研究与荧光团标记的人淀粉样蛋白共定位和吞噬的光学成像 小鼠的PMφ。AL淀粉样蛋白吞噬功能的定量显著改善 CAR将通过与CAR阴性的Mφ和使用11-1F4的淀粉样蛋白直接操作化进行比较来评估。 淀粉样蛋白导向的CAR-PMφ是治疗AL及其相关系统的一种新方法 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 淀粉样变性治疗的清除模式。鉴于这些研究的积极数据，我们希望 淀粉样蛋白导向的CAR-P技术可以在有效性和实用性方面进行优化，并转化为临床 通过提高生活质量和延长患者生存时间，为患者提供有意义的好处。

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.