Preclinical Diagnostic Imaging of Amyloid

描述(申请人提供)：淀粉样蛋白与一组通常致命的遗传性和散发性蛋白质错误折叠障碍有关，其特征是纤维和硫酸乙酰肝素蛋白多糖在重要器官和组织中沉积。虽然最常见的表现是阿尔茨海默病患者，但在美国，外周(非脑性)淀粉样病的发病率估计为每年约5300例，超过急性淋巴细胞白血病(每年约4000例)。外周淀粉样变的治疗选择有限，主要集中在减少淀粉样蛋白的合成，例如，使用大剂量化疗，目前正在进行31项治疗AL、AA和ATTR淀粉样变的新试验(Clinicaltrials.gov)。不幸的是，在美国还没有方法可以直接可视化这些抗淀粉样蛋白治疗的反应，也没有方法确定淀粉样蛋白沉积的范围和严重程度或患者的靶器官。这类信息可以通过使用淀粉样蛋白特异性放射性示踪剂进行成像来获得。因此，迫切需要确定以淀粉样蛋白为靶点的全身成像示踪剂，以便在临床内和作为临床试验的一部分，用于监测疾病进展和治疗反应。这项建议的目的是瞄准淀粉样生物标记物硫酸乙酰肝素蛋白多糖，并测试抗体衍生的和与所有已知的淀粉样沉淀物中的这一成分特异结合的多肽示踪分子。我们的方法是基于这样一个事实，即所有淀粉样沉积都含有硫酸乙酰肝素蛋白多糖，含量高达每克病变组织250 5g。我们已经开始测试一组已知与硫酸肝素结合的抗体衍生蛋白(ScFv)，以确定它们与淀粉样蛋白沉积中的硫酸肝素结合的能力。我们已经证明，尽管在一些正常组织中有硫酸肝素的表达，但对过硫酸肝素的ScFv在体内成功地成像了淀粉样蛋白。还将产生和测试新型的硫酸乙酰肝素结合肽。在体外，将通过表面等离子激元共振和ELISA对淀粉样蛋白反应性单链抗体和多肽进行表征，以确定那些具有平衡结合亲和力的单链抗体适合于患者进行成像。小动物SPECT、PET和CT成像将用于比较现有的和新型的硫酸乙酰肝素结合示踪剂的共存情况，并确定那些具有可接受的(3：1)目标与背景比率的示踪剂。我们将使用已建立的全身性外周(AA)淀粉样变性的小鼠模型，该模型概括了人类淀粉样病的许多方面，包括蛋白多糖的沉积，这增加了我们将识别出有利于转化为临床的淀粉样变性成像示踪剂的可能性。鉴定和验证在组织淀粉样蛋白中发现的特异性结合硫酸乙酰肝素蛋白多糖的分子将为这种毁灭性疾病的患者的临床评估提供新的试剂。公共卫生相关性：外周淀粉样变性是一种蛋白质错误折叠状态，与2型糖尿病、慢性炎症性疾病，如结核病和类风湿性关节炎，以及某些B细胞恶性肿瘤有关。目前，美国还没有可用的方法来检测外周淀粉样变性患者中淀粉样蛋白沉积的程度，也没有方法可以监测其进展，或记录治疗后它们的清除情况。这类信息可以通过使用淀粉样蛋白特异性放射性示踪剂进行成像来获得。为此，我们的目标是瞄准淀粉样生物标记物硫酸乙酰肝素蛋白多糖，并测试抗体衍生的和肽示踪分子，这些分子专门与所有已知的淀粉样沉积的这种成分结合，用于全身成像，可用于监测疾病进展和治疗反应。

DESCRIPTION (provided by applicant): Amyloid is associated with a diverse group of often fatal hereditary and sporadic protein misfolding disorders, characterized by the deposition of fibrils and heparan sulfate proteoglycan in vital organs and tissues. Although the most common manifestation is in patients with Alzheimer's disease, the incidence of peripheral (non-cerebral) amyloid diseases in the USA, is estimated to be ~ 5300 per yr which exceeds that for acute lymphocytic leukemia (~ 4,000 per yr). Treatment options for peripheral amyloidosis are limited and focus on reducing synthesis of the amyloidogenic protein e.g., with high dose chemotherapy and there are currently 31 new trials for treating AL, AA, and ATTR amyloidosis underway (clinicaltrials.gov). Unfortunately, there are no methods available in the USA to visualize response to these anti-amyloid therapies directly, nor to determine the extent and severity of amyloid deposits or the target organ in patients. This kind of information can be obtained by imaging, using an amyloid-specific radiotracer. There exists therefore an urgent need to identify tracers that target amyloid for whole body imaging that can be used for monitoring disease progression and response to therapy both within the clinic and as part of clinical trials. The aim of this proposal is to target the amyloid biomarker heparan sulfate proteoglycan and test antibody-derived and peptide tracer molecules that bind specifically to this constituent of all known amyloid deposits. Our approach is based on the fact that all amyloid deposits contain heparan sulfate proteoglycan at levels as high as 250 5g per gram of diseased tissue. We have begun testing a panel of antibody-derived proteins (scFv) known to bind heparan sulfate, for their ability to bind heparan sulfate in amyloid deposits. We have shown that scFvs to hypersulfated heparin sulfate successfully imaged amyloid in vivo even though heparan sulfate was expressed in some normal tissues. Novel heparan sulfate-binding peptides will also be generated and tested. In vitro characterization of the amyloid-reactive scFv and peptides will be performed by surface plasmon resonance and ELISA to identify those scFv with equilibrium binding affinity of < 1 5M - suitable for imaging in patients. Small animal SPECT, PET, and CT imaging will be used to compare the co-localization of the available and novel heparan sulfate- binding tracers and identify those with acceptable (3:1) target to background ratios. We will use an established murine model of systemic peripheral (AA) amyloidosis that recapitulates many aspects of human amyloid disease including the deposition of proteoglycans which enhances the probability that we will identify tracers for imaging amyloidosis that translate favorably into the clinic. Identifying and validating molecules that specifically bind heparan sulfate proteoglycan found in tissue amyloid will provide new reagents for the clinical evaluation of patients with this devastating disease. PUBLIC HEALTH RELEVANCE: Peripheral amyloidosis is a protein-misfolding condition associated with type 2 diabetes, chronic inflammatory disorders such as tuberculosis and rheumatoid arthritis, as well as certain B-cell malignancies. Currently here are no methods available in the USA that can detect the extent of amyloid deposits in patients with peripheral amyloidosis, nor to monitor their progression, or document their removal in response to therapy. This kind of information can be obtained by imaging, using an amyloid-specific radiotracer. To this end, we aim to target the amyloid biomarker heparan sulfate proteoglycan and test antibody-derived and peptide tracer molecules that bind specifically to this constituent of all known amyloid deposits for the purpose of whole body imaging that can be used to monitor disease progression and response to therapy.