Releasable Antibodies for Multiplexed Analysis of Cancer Biomarkers

描述（由申请人提供）：本提案旨在克服当前和新兴的多路复用技术的局限性，这些技术对改善癌症诊断和治疗至关重要。这些技术利用经典抗体进行生物标记物检测，因此受到抗体培养物种的稀缺性、位阻、苛刻的多路复用条件以及细胞或组织样品的永久性修饰的阻碍。为了克服这些限制，同时能够对活细胞进行多重分析，GE将开发具有可控亲和力的不稳定抗体。具体来说，为了演示目的，将对现有的抗HER2抗体进行修饰，并以不同程度的修饰制备所得的偶联物，并通过UV-vis光谱、LDS-PAGE和MALDI-TOF-MS进行表征。然后将利用荧光显微镜评估这些偶联物与固定SKOV-3细胞颗粒结合HER2的能力，并通过表面等离子体共振（Biacore）对结合动力学进行量化。所得信息将用于制备改进的抗体偶联物，以验证该技术的多路复用能力。具体来说，HER2和Ki67生物标志物的序列检测将在临床相关的人类乳腺癌样本中使用来自单一物种的一抗进行。这项工作的具体目标将是制备和表征修饰的抗her2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在临床相关组织样本上的多路复用能力。该技术的成功开发将允许与来自同一物种的一抗进行无限多路复用；同一组织样品中多个紧密间隔的生物标志物的检测和定量；生物标志物空间信息评价；生物标志物之间、与疾病进展和治疗反应的相关性；除了固定组织，还有机会对活细胞进行多重分析。

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.