Releasable Antibodies for Multiplexed Analysis of Cancer Biomarkers

描述(由申请人提供)：这项提案旨在克服当前和新兴的多路技术的局限性，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，因此受到抗体产生物种稀缺、空间障碍、苛刻的多重条件以及细胞或组织样本的永久修饰的阻碍。为了克服这些限制，同时能够对活细胞进行多重分析，GE将开发亲和力可控的不稳定抗体。具体地说，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得到的结合物，并用UV-Vis光谱、LDS-PAGE和MALDI-TOF-MS对其进行表征。这些偶联物将通过荧光显微镜评估它们使用固定的SKOV-3细胞颗粒与HER2结合的能力，结合动力学将通过表面等离子体共振(Biacore)来定量。所得信息将用于制备改进的抗体结合物，以验证该技术的多路传输能力。具体地说，将使用单一物种的初级抗体在临床相关的人类乳腺癌样本中进行HER2和Ki67生物标记物的顺序检测。这项工作的具体目标将是制备和表征修饰的抗HER2抗体库，识别和优化铅抗体结合物，并验证铅抗体结合物在临床相关组织样本上的多重能力。拟议技术的成功开发将允许与来自同一物种的初级抗体进行无限多重；在同一组织样本中检测和量化多个紧密相距的生物标记物；评估生物标记物的空间信息；生物标记物之间的相互关联、疾病进展和治疗反应；以及除固定组织外，对活细胞进行多重分析的机会。 与公共卫生相关：以多重方式筛选组织样本或单个固定或活细胞中的生物标记物的能力对于增强癌症诊断和开发更有针对性的治疗方法至关重要。拟议的项目将通过对针对疾病生物标记物的抗体进行化学修饰来实现这一点，以实现可控的亲和力，这将使有效结合和释放成为可能。这些抗体将保持组织或细胞样本的完整性，同时实现潜在的无限多重能力，而不会由于有限的抗体种类可获得性或重叠的生物标记表位而受到限制。

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.