Identification of Immune Selected Breast Cancer Antigens

描述(由申请人提供)： 免疫依赖反应在定义非我或异常抗原提呈时是有选择性的。不幸的是，针对人类癌症抗原的长期抗体产生是有限的。在一种创新和新颖的方法中，我们开发了一种方法，利用肿瘤引流淋巴中的初级免疫反应来提供定义源自乳腺癌的生物活性肿瘤抗原的手段。这一新的方法结合了一系列步骤来协调低复杂性抗体cDNA文库的构建和蛋白质生产，用于通过灵敏的抗体微米级“抗原陷阱”分析和LC-MS/MS抗原鉴定来鉴定肿瘤抗原。识别出的肿瘤抗原可以用生化、免疫学和分子方法来验证为潜在的肿瘤抗原。应用的方法是基于中等吞吐量平台的设计，以快速评估来自同一患者的匹配的淋巴和肿瘤样本。该项目应用创新技术证明：a)肿瘤引流淋巴结对变异的乳腺癌抗原具有免疫反应，并在增殖的B细胞生发中心产生抗原依赖型体细胞突变；b)重组VH和/或VHV1/VK scFv蛋白合成的体细胞高突变抗体的抗原结合区域可特异性识别和识别乳腺癌抗原；c)已确定的抗原可被证实为乳腺癌亚型的诊断。该R21应用程序建议扩展和改进我们的方法，以：1)在更大的患者群体中确定肿瘤抗原免疫选择的多样性和有效性，2)扩大我们生产具有适当结构和抗原结合的抗体分子的能力，以及3)开发方法，将合成的抗体蛋白质结合到高灵敏度的分析中，以筛选评估抗原作为诊断生物标志物的潜力所需的原发癌症、组织材料和/或生物液。

DESCRIPTION (provided by applicant): Immune-dependent responses are selective in defining non-self or aberrant antigen presentation. Unfortunately, long-term antibody production to human cancer antigens is limited. In an innovative and novel approach, we have developed a method to utilize primary immune reactions in tumor draining lymph nodes to provide the means to define biologically active tumor antigens originating from breast cancers. This novel approach combines a series of steps to coordinate the construction of low complexity antibody cDNA libraries and protein production that are used to identify tumor antigens using sensitive antibody microscale "antigen-trap" assays followed by LC-MS/MS antigen identification. Tumor antigens identified can then be verified as potential tumor antigens using biochemical, immunological and molecular methodologies. The methodologies applied are medium throughput platform based designed to rapidly evaluate matched lymph node and tumor samples from the same patient. This project applies innovative technologies that demonstrate that: a) tumor draining lymph nodes are immuno-reactive to aberrant breast cancer antigens and produce antigen-dependent somatic hypermutation in proliferative B-cell germinal centers, b) antigen binding domains of somatic hypermutated antibodies synthesized as recombinant VH and/or VHVl/Vk ScFv proteins can specifically recognize and identify breast cancer antigens, and c) antigens identified can be verified as diagnostic for breast cancer sub-phenotypes. This R21 application proposes to expand and refine our methodologies to: 1) determine the diversity and effectiveness of immune-selection of tumor antigens in a larger patient population, 2) expand our ability to produce antibody molecules with appropriate structure and antigen binding, and 3) develop methodologies to incorporate antibody proteins synthesized into highly sensitive assays that can screen primary cancer, histological material and/or biological fluids necessary to evaluate the potential of antigens as diagnostic biomarkers.