Granulin specific monoclonal antibodies to investigate their expression and role

摘要 美国有超过700万的痴呆症患者(约占总人口的3%)，其中约5%是额颞部的 痴呆症(FTD)。最近有研究表明，美国5%-10%的FTD是由基因突变引起的 原颗粒蛋白(PGRN)基因。这伴随着患者体内生物体内PGRN水平的50%下降 体液。PGRN是一个由593个氨基酸组成的生长和存活因子。PGRN的克隆和序列分析表明，它 包含一个17个氨基酸的信号肽用于分泌和7个半6 kDa GRN重复，具有独特的 串联重复双半胱氨基富含颗粒蛋白(GRN)基序。6个KDA GRN是通过以下处理生成的 弹性酶刺激的蛋白水解性裂解和分泌性白细胞蛋白酶抑制剂阻断的PGRN 斯尔皮。最近的证据表明，PGRN和GRN都可能直接影响神经退行性疾病 过程中，如阿尔茨海默氏症、帕金森氏病和某些类型的FTD。有人建议说， PGRN和GRN水平的平衡对于神经元保护/退化是重要的，因为 发炎。因此，对PGRN和GRN水平的测量可能有助于了解正常 而不是大脑中与疾病相关的过程。目前，人们对其功能和功能知之甚少 特定GRN在脑中的表达与PGRN的比较及其可能的联系机制 PGRN和GRN对这些疾病的作用。因此，开发特定的有效检测系统以促进 神经退行性疾病中的PGRN/GRN正向场PI是PGRN研究领域公认的专家 特别是在癌症方面，并已开发出经临床验证的高质量单抗和检测方法，以 检测患者组织和生物液中的PGRN。她的PGRN分析和试剂也用于 与两个卓越的神经退行性疾病中心合作研究。然而，没有 测量GRN分子的可靠、灵敏和特异的分析方法，从而阻碍了对其作用的了解 GRN在神经退行性疾病中的作用。PI正在请求对此R03应用程序的支持，以开发 针对GRN的单抗组合。该方法将针对新的裂解位点的 7个不同的GRN，以制备适用于这7个GRN的夹心ELISA法的单抗。具体地说，它 建议：1)研制适用于夹心ELISA7种GRN分子的高亲和力单抗 在生物样品中的检测：2)开发标准化的实验室检测方法，专门检测7种GRN 在浓度低于100pg/ml且不受PGRN干扰的生物液体中。这些研究 将是重要的，因为为7个GRN中的每一个开发有效的夹心ELISA试剂盒，它将使 研究人员调查脑脊液和其他生物中各种GRN和PGRN的存在和比例 来自FTD等神经退行性疾病患者的样本。提供可检测测量的工具 阐明这些重要生物制品的作用也可能有助于揭示 将它们用作疾病或治疗监测的治疗靶点或替代标记物。

Abstract Over 7 million cases of dementia exist in the US (~3% of the population) and of these ~5% are frontotemporal dementia (FTD). It has been demonstrated recently that 5-10% of all FTD in the US are caused by mutations in the Progranulin (PGRN) gene. This is accompanied with a 50% reduction of PGRN level in patients' biological fluids. PGRN is a 593 amino-acid growth and survival factor. Cloning and sequencing of PGRN showed that it contains a 17 amino-acid signal peptide for secretion and seven and a half 6 kDa grn repeats with a unique tandem repeat double cysteinyl rich granulin (grn) motif. The 6 Kda Grns are generated by processing of PGRN by a proteolytic cleavage stimulated by elastase and blocked by secretory leucocyte protease inhibitor SLPI. Recent evidence suggests that both PGRN and grns may directly influence neurodegenerative diseases process, such as Alzheimer's disease, Parkinson's disease and certain types of FTD. It has been suggested that the balance of PGRN and grn levels is important for neuron protection/degradation as a result of inflammation. Accordingly, the measurement of PGRN and grn levels may shed light on the balance of normal versus disease related processes in the brain. Currently, relatively little is known about the function and expression of specific grns in the brain when compared to PGRN and about the possible mechanisms linking PGRN and grn to these diseases. Thus, it is critical to develop specific validated assay systems to advance the PGRN/grn field forward in the neurodegenerative disease. The PI is a recognized expert in PGRN research particularly in cancer and has developed clinically validated high quality monoclonal antibodies and assays to measure PGRN in tissue and biological fluids in patients. Her PGRN assays and reagents are also used in collaborative studies with two centers of excellence in neurodegenerative disease. However, there are no reliable, sensitive and specific assays to measure grn molecules, thereby hampering understanding of the role of grns in neurodegenerative diseases. The PI is requesting support in this RO3 application to develop a portfolio of monoclonal antibodies specific to grn. The approach will be to target the neo-cleavage sites of the seven different grns to develop mAbs suitable for sandwich ELISA assay for each of the 7 grns. Specifically, it is proposed to: 1) Develop high affinity mAbs specific to each of 7 grn molecule suitable for sandwich ELISA detection in biological samples: 2) Develop standardized laboratory assays for specifically detecting the 7 grns in biological fluids at concentrations of less than 100 pg/ml and without interference by PGRN. These studies will be important as the development of validated sandwich ELISA kits for each of the 7 grns, it will enable researchers to investigate the presence and the ratio of various grns and PGRN in CSF and other biological samples from patients with neurodegenerative diseases such as FTD. Providing tools that can detect measure and elucidate the role of these important biological products may also be useful in revealing the potential to use them as therapeutic targets or surrogate markers for disease or therapy monitoring.