Processed Antigen Characterization by Mass Spectrometry

项目摘要 人体内的细胞通过降解细胞向免疫系统传递其健康状态 蛋白质和呈现片段的每一个在细胞表面的结合类MHC蛋白。 受过适当教育的细胞毒性T淋巴细胞(CTL)(CD8+T细胞)与I类MHC分子结合在一起 细胞表面，对呈现的蛋白质片段(肽)进行采样，并杀死表达 病毒、细菌和寄生虫感染、组织移植和细胞移植所致的新多肽 变形(癌症)。由于细胞信号通路的失调是癌症的标志之一，我们 假设从这些途径产生的I类MHC磷酸肽应该是很好的 用于癌症免疫治疗的候选药物。初步鉴定的I类MHC磷酸肽 对白血病、黑色素瘤和结直肠癌的研究引发了预先存在的、中央记忆的T细胞回忆 多名健康献血者的反应。中枢记忆回忆对磷酸肽抗原的反应是 在一些白血病患者中不存在，并与临床结果相关。响应将在以下时间恢复 异基因干细胞移植。这些结果有力地表明，I类MHC磷酸肽是 肿瘤是人类免疫监视的靶点，因此很可能是免疫治疗的候选对象。 癌症。其中三种已发现的磷酸肽将作为黑色素瘤I期临床试验的对象。 今年春天晚些时候。这里建议进行额外的研究，以完成我们对黑色素瘤、白血病和 结直肠癌，并开始一项工作，以表征I类MHC磷酸酯 食道癌、肝癌和卵巢癌。将进行更多研究，以确定以下曲目 由处方药或环境制剂诱导的改变的I类或II类自体多肽 导致威胁生命的自身免疫性疾病。 还提出了开发新的质谱学技术，将有助于接近完成 完整治疗性抗体和其他蛋白质在高Femtomole水平上的氨基酸序列覆盖率 在层析时间刻度上。这将通过一种组合蛋白质化学物质来完成 衍生化、对现有仪器硬件的更改、增强内部离子-离子化学的性能 仪器，以及对仪器控制软件的修改。这种方法应该会加速 开发针对多种疾病的治疗性抗体，并促进对 在同一蛋白质上包含多个翻译后修饰的蛋白质和蛋白质复合体 分子。

Project Summary Cells in the human body communicate their health status to the immune system by degrading cellular proteins and presenting fragments of each on the cell surface in association class I MHC proteins. Appropriately educated, cytotoxic T-lymphocytes (CTL) (CD8+ T-cells) bind to the class I MHC molecules on the cell surface, sample the protein fragments (peptides) being presented and kill those cells that express new peptides as a result of viral, bacterial and parasitic infection, tissue transplantation and cellular transformation (cancer). Since dysregulation of cell signaling pathways is one of the hallmarks of cancer, we hypothesized that class I MHC phosphopeptides that result from these pathways should be excellent candidates for use in the immunotherapy of cancer. Class I MHC phosphopeptides identified in preliminary work on leukemia, melanoma, and colorectal cancers elicit pre-existing, central-memory, T-cell-recall responses in multiple, healthy blood donors. Central memory recall responses to phosphopeptide antigens is absent in some leukemia patients and correlates with clinical outcome. The response is restored following allogenic stem cell transplantation. These results suggest strongly that class I MHC phosphopeptides are tumor targets of immune surveillance in humans and, therefore, are likely candidates for immunotherapy of cancer. Three of the discovered phosphopeptides will be the subject of a phase I clinical trial on melanoma later this spring. Proposed here is additional research to complete our studies on melanoma, leukemia, and colorectal cancer and to begin an effort to characterize class I MHC phosphopetides presented on esophageal, liver and ovarian cancer. Additional research will be conducted to identify the repertoire of altered class I or class II self-peptides that are induced by prescription drugs or environmental agents and lead to life threatening autoimmune disease. Also proposed is the development novel mass spectrometry technology that will facilitate near complete amino acid sequence coverage of intact therapeutic antibodies and other proteins at the high femtomole level on a chromatographic time scale. This will be accomplished by a combination protein chemical derivatization, changes to existing instrument hardware, enhanced performance of ion-ion chemistry inside the instrument, and modifications to the instrument control software. This approach should accelerate the development of therapeutic antibodies for multiple diseases and also facilitate complete characterization of proteins and protein complexes that contain multiple posttranslational modifications on the same protein molecule.