ScFv Piezoimmunosensor Detection of Therapeutic Antibodies in Human Serum

描述（由申请人提供）：治疗性单克隆抗体（mabs）在治疗人类疾病（如癌症和类风湿性关节炎）方面表现出巨大的潜力。超过300种抗体正在进行临床开发，2915项涉及抗体的临床研究正在进行。治疗性抗体的效果是浓度依赖性的。然而，在一些患者中，治疗性抗体可以迅速从体内消除，对患者几乎没有好处。如果患者本可以从替代疗法中获益，但没有给予替代疗法，那么患者的疾病可能会在治疗性抗体治疗过程中恶化或危及生命。然而，如果医生可以利用化验结果轻易地确定治疗性抗体浓度，那么医生就可以调整治疗以使患者受益。传统的免疫测定（如酶联免疫吸附测定或elisa）通常结合抗原来检测血清样品中的抗原特异性抗体。然而，许多治疗性抗体识别的抗原可能不稳定，难以处理或获得昂贵。因此，它们不能总是用来建立一个简单、稳定、可重复的试验。治疗性抗体已被设计或开发成具有正常人类抗体的大部分（如果不是全部的话）免疫学特征，因此它们可以用于人类而不会引起不良的免疫反应。由于它们与正常的人抗体相似，在不使用互补抗原的情况下，很难在人血清中检测和定量。目前，还没有快速、简单、高灵敏度、廉价的方法来检测人类血清样本中的大多数治疗性抗体。我们已经成功开发了一种非常简单，但功能非常强大的基于scfv的压电免疫传感器检测平台，用于检测血清样品中的独特抗体。在本项目的目标1中，我们建议利用一个庞大的（约29亿个成员）噬菌体显示抗体库来选择稳定的针对独特治疗抗体的重组抗体。治疗性抗体特异性scFv将用于替代Aim 2的传统抗原，以开发压电免疫传感器，以检测和量化人血清样本中的治疗性抗体。压电免疫传感器将scFv识别抗原（即治疗性抗体）的高特异性和稳定性与低成本、无标签石英晶体微平衡传感器的一步读出相结合。曾博士是一名分析化学家，默诺博士是一名免疫学家，他们将进行拟议的研究。通过他们的协同努力，该团队将创建并认证一个快速、准确、高灵敏度和特异性、稳定、低成本的诊断分析平台，可用于检测和量化人类血清样本中的治疗性抗体（例如贝伐单抗、西妥昔单抗、帕尼单抗和曲妥珠单抗）。

DESCRIPTION (provided by applicant): Therapeutic monoclonal antibodies (McAbs) exhibit tremendous potential to treat human disease such as cancer and rheumatoid arthritis. More than 300 antibodies are undergoing clinical development and 2915 clinical studies involving antibodies are being carried out. The effect of therapeutic antibodies is concentration dependent. However, in some patients, therapeutic antibodies can be rapidly eliminated from the body and will be of little benefit to the patient. If a patient could have benefited from, but was not given an alternative therapeutic, then the patient's disease could have worsened or become life threatening during the course of therapeutic antibody treatment. However, if a physician can use the results of an assay to readily determine therapeutic antibody concentration, then the physician can adjust treatment to the benefit of the patient. Traditional immunoassays (e.g. enzyme-linked immunosorbant assays or ELISAs) typically incorporate antigens to detect antigen-specific antibodies in serum samples. However, the antigens recognized by many therapeutic antibodies can be unstable, difficult to work with or expensive to acquire. As such, they cannot always be used to build a simple, stable, reproducible assay. Therapeutic antibodies have been engineered or developed to assume most, if not all of the immunological features of a normal human antibody, so that they can be used in humans without eliciting an adverse immune response. Due to their similarities to normal human antibodies, they can be difficult to detect and quantify in human serum without the use of complementary antigens. At present, rapid, simple, highly sensitive, inexpensive assays to detect most of these therapeutic antibodies in human serum samples are not readily available. We have successfully developed a very simple, but extremely powerful scFv-based piezoimmunosensor assay platform to detect unique antibodies in serum samples. We propose in Aim 1 of this project to utilize a large (~2.9 billion member) phage-displayed antibody library to select for stable scFv recombinant antibodies specific for unique therapeutic antibodies. The therapeutic antibody-specific scFv will be used in lieu of traditional antigens for Aim 2 to develop piezoimmunosensors to detect and quantify therapeutic antibodies in human serum samples. The piezoimmunosensors will combine the high specificity and stability of scFv recognition of antigen (i.e. therapeutic antibody) with the low cost, one-step readout of non-label Quartz Crystal Microbalance transducers. Dr. Zeng, an analytical chemist, and Dr. Mernaugh, an immunologist, will carry out the proposed studies. Through their synergistic efforts, this team will create and certify a fast, accurate, highly sensitive and specific, stable, low cost diagnostic assay platform that can be used to detect and quantify therapeutic antibodies (e.g. Bevacizumab, Cetuximab, Panitumumab and Trastuzumab) in human serum samples. PUBLIC HEALTH RELEVANCE: The proposed research will focus on developing scFv recombinant antibodies and scFv-based piezoimmunosensor assays for use in detecting therapeutic antibodies in human serum samples. At present, rapid, simple, highly sensitive, inexpensive assays to detect most of therapeutic antibodies in human serum samples are not readily available. Since the proposed assay will be more sensitive than traditional immunoassay (e.g. ELISAs or whole antibody immunosensors) and can rapidly and accurately detect therapeutic antibodies in humans using a few drops of human serum and inexpensive instrumentation, a physician will be able to quickly determine if the concentration of therapeutic antibody in patients is sufficient or if therapy must be changed to benefit the patient.