Releasable Antibodies for Multiplexed Analysis of Cancer Biomarkers

描述（由申请人提供）：该提案旨在克服对改善癌症诊断和治疗至关重要的当前和新兴多重技术的局限性。这些技术利用经典抗体进行生物标志物检测，并且因此受到抗体产生的物种的稀缺性、空间位阻、苛刻的多重条件和细胞或组织样品的永久修饰的阻碍。为了克服这些限制，同时实现活细胞的多重分析，GE将开发具有可控亲和力的不稳定抗体。具体地，为了证明的目的，将修饰现有的针对HER 2的抗体，并且将以不同的修饰程度制备所得的缀合物，并通过紫外-可见光谱表征，LDS-PAGE和MALDI-TOF-MS。然后通过荧光显微镜使用固定的SKOV-3细胞沉淀物评价这些缀合物结合HER 2的能力，并通过表面等离子体共振（比亚科雷）。所得到的信息将用于制备改进的抗体缀合物，以验证该技术的多路复用能力。具体而言，将使用来自单一物种的一抗在临床相关的人乳腺癌样本中进行HER 2和Ki 67生物标志物的连续检测。这项工作的具体目的是制备和表征修饰的抗HER 2抗体库，鉴定和优化先导抗体偶联物，并验证先导抗体偶联物对临床相关组织样本的多重作用能力。所提出的技术的成功开发将允许与来自相同物种的一抗的无限多路复用;检测和定量相同组织样品中的多个紧密间隔的生物标志物;评估生物标志物空间信息;生物标志物彼此、疾病进展和治疗反应的相关性;以及除了固定组织之外对活细胞进行多路复用分析的机会。 公共卫生相关性：以多重方式筛选组织样本或单个固定或活细胞的生物标志物的能力对于增强癌症诊断和开发更具靶向的治疗至关重要。拟议的项目将通过化学修饰针对疾病生物标志物的抗体来实现这一目标，以实现可控的亲和力，从而实现有效的结合和释放。这些抗体将保持组织或细胞样品的完整性，同时能够实现潜在的无限多路复用能力，而不受由于有限的抗体种类可用性或重叠的生物标志物表位而造成的限制。

DESCRIPTION (provided by applicant): This proposal seeks to overcome the limitations of current and emerging multiplexed technologies that are critical to improving cancer diagnosis and treatment. These technologies utilize classical antibodies for biomarker detection, and as such are hampered by scarcity of species in which the antibodies are raised, steric hindrance, harsh multiplexing conditions, and permanent modification of the cell or tissue sample. To overcome these limitations and simultaneously enable multiplex analysis of living cells, GE will develop labile antibodies with controllable affinity. Specifically, for demonstration purposes, existing antibodies against HER2 will be modified and the resulting conjugates will be prepared at varying degrees of modification and characterized by UV-vis spectroscopy, LDS-PAGE and MALDI-TOF-MS. These conjugates will then be evaluated for their capability to bind HER2 using fixed SKOV-3 cell pellets by fluorescence microscopy and the binding kinetics will be quantified via surface plasmon resonance (Biacore). The resulting information will be used to prepare improved anti- body conjugates for validation of the technology's multiplexing capabilities. Specifically, sequential detection of HER2 and Ki67 biomarkers will be performed in clinically-relevant human breast cancer samples using primary antibodies from a single species. The specific aims of the work will be to prepare and characterize a library of modified anti-HER2 antibodies, identify and optimize lead antibody conjugates, and to validate the multiplexing capabilities of the lead antibody conjugates on clinically-relevant tissue samples. Successful development of the proposed technology will allow unlimited multiplexing with the primary antibodies from the same species; detection and quantification of multiple closely-spaced biomarkers in the same tissue sample; assessment of biomarker spatial information; correlation of biomarkers to each other, to disease progression, and to treatment response; and the opportunity to perform multiplex analysis on living cells in addition to fixed tissue. PUBLIC HEALTH RELEVANCE: The ability to screen tissue samples or individual fixed or living cells for biomarkers in a multiplexed fashion is critical for enhanced cancer diagnostics and the development of more targeted therapies. The proposed project will achieve this by chemically modifying antibodies targeted against disease biomarkers to achieve controllable affinity, which will enable efficient binding and release. These antibodies will preserve the integrity of the tissue or cell sample while enabling potentially unlimited multiplexing capabilities without restrictions due to limited antibody-species availability or overlapping biomarker epitopes.