Identification of Immune Selected Breast Cancer Antigens

描述（由申请人提供）： 免疫依赖性应答在定义非自身或异常抗原呈递方面是选择性的。不幸的是，针对人类癌症抗原的长期抗体产生是有限的。在一个创新的和新的方法，我们已经开发出一种方法，利用原发性免疫反应在肿瘤引流淋巴结提供的手段来定义生物活性肿瘤抗原来源于乳腺癌。这种新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，所述蛋白质生产用于使用灵敏的抗体微量“抗原捕获”测定法随后进行LC-MS/MS抗原鉴定来鉴定肿瘤抗原。然后可以使用生物化学、免疫学和分子方法学将鉴定的肿瘤抗原验证为潜在的肿瘤抗原。所应用的方法是基于中等通量平台的，旨在快速评价来自同一患者的匹配淋巴结和肿瘤样品。该项目采用的创新技术证明：a）肿瘤引流淋巴结对异常乳腺癌抗原具有免疫反应性，并在增殖性B细胞生殖中心产生抗原依赖性体细胞超突变，B）合成为重组VH和/或VHV 1/Vk ScFv蛋白的体细胞超突变抗体的抗原结合结构域可特异性识别和鉴定乳腺癌抗原，和c）所鉴定的抗原可被验证为乳腺癌亚表型的诊断。此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的免疫选择的多样性和有效性，2）扩大我们产生具有适当结构和抗原结合的抗体分子的能力，和3）开发将合成的抗体蛋白质掺入可以筛选原发性癌症的高灵敏度测定中的方法，组织学材料和/或生物流体，这些是评价抗原作为诊断生物标志物的潜力所必需的。

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.