Flex-Hinge Bispecific Antibodies for Ultra-Sensitive Detection of Beta Amyloid Species in Plasma

项目摘要/摘要 阿尔茨海默病(AD)是一种毁灭性的进行性神经退行性疾病，估计有11%的人受到影响 65岁以上的美国人和33-50%的85岁以上的人。阿尔茨海默病的最初病理征象--β-内毒素积聚 大脑中的淀粉样蛋白(Aβ)斑块，通常是认知障碍的先兆。虽然这些斑块可以用β成像 PET脑扫描或通过分析脑脊液中的Aβ多肽进行检测，这些方法成本高昂，具有侵入性和 不方便。最近，临床研究表明，疗法在治疗阿尔茨海默病方面可能更有效 在疾病的早期阶段，即抗A-β疗法可以从大脑中去除A-β，需要一种简单、廉价的 对AD发病和进展的诊断血液测试甚至更大。很难开发出一种基于 Aβ，部分原因是血液中Aβ多肽浓度较低，而其他蛋白质浓度较高。我们 已经通过开发方法来连接抗原结合区域(Fabs)来解决这个蛋白质设计问题 抗体珠子使用灵活和可扩展的非肽基铰链。珠子上的柔性连接的Fabs能够 通过在双手中协同结合，有效地捕获血液中极低水平的Aβ(307attomoles) 时尚。这些弹性铰链™Fab珠子，结合MALDI-TOF质谱仪，先前揭示了 未知形式的β，包括一种新的血浆中作为精确定性和定量替代的β生物标志物 对于脑Aβ正电子发射计算机断层扫描，与AD的分期相关。在第一阶段SBIR中，我们将在这些结果的基础上改进 两个关键方法：(1)我们将生产弹性铰链™双特异性抗体微珠，而不是使用弹性铰链™Fab微珠 我们希望通过改善协作性和减少变异性，具有更高的敏感性和实用性，以及(2) 我们将转向Orbitrap技术，与MALDI相比，该技术对Aβ多肽具有更高的灵敏度和分辨率。 我们将使用这些弹性铰链™双特异性抗体微珠和Orbitrap技术来检查来自 瑞典生物指纹研究证实新的β生物标记物的实用性并确定额外的β生物标记物(S) 血浆。第二阶段研究将使用更大的样本集来评估该分析。最终，我们打算将我们的测试变成一个工具包 它将被广泛使用，并成为监测AD的常规方法，就像用胆固醇测试进行监测一样 心血管疾病。

PROJECT SUMMARY/ABSTRACT Alzheimer’s disease (AD) is a devastating, progressive neurodegenerative disease affecting an estimated 11% of Americans over 65 years of age and 33-50% over 85 years of age. The initial pathological sign of AD, the build-up of beta amyloid (Aβ) plaques in the brain, often precedes impairment of cognition. Although these plaques can be imaged by Aβ PET brain scans or detected via analysis of Aβ peptides in the cerebrospinal fluid, these methods are costly, invasive and inconvenient. Recently, with clinical studies indicating that therapeutics may be more effective in treating AD at the earliest stages of the disease and that anti-Aβ therapy can remove Aβ from the brain, the need for a simple, inexpensive diagnostic blood test for AD onset and progression is even greater. It has been difficult to develop a blood test based on Aβ, in part because of the low concentration of Aβ peptides in blood and the high concentration of other proteins. We have approached this protein design problem by developing methods to link the antigen binding domains (Fabs) of antibodies to beads using flexible and extendible non-peptidyl hinges. The flexibly-linked Fabs on the beads are able to efficiently capture exceptionally low levels of Aβ (307 attomoles) in blood by binding cooperatively in a two-handed fashion. These Flex-Hinge™ Fab beads, combined with MALDI-TOF mass spectrometry, have revealed previously unknown forms of Aβ including a novel Aβ biomarker in plasma that acts as a precise qualitative and quantitative proxy for brain Aβ PET imaging, correlating with the stages of AD. In this Phase I SBIR, we shall improve upon these results in two key ways: (1) instead of using Flex-Hinge™ Fab beads, we shall produce Flex-Hinge™ Bispecific Antibody beads that we expect to have even greater sensitivity and utility through improved cooperativity and reduced variability, and (2) we shall move to Orbitrap technology, which has greater sensitivity and resolution for Aβ peptides compared to MALDI. We shall use these Flex-Hinge™ Bispecific Antibody beads and Orbitrap technology to examine clinical samples from the Swedish BioFINDER study to confirm the utility of the novel Aβ biomarker and identify additional Aβ biomarker(s) in plasma. Phase II studies shall evaluate this assay with larger sample sets. Ultimately, we intend to turn our test into a kit that will be broadly available and become routine for monitoring AD, much as cholesterol testing is used for monitoring cardiovascular disease.