Processed Antigen Characterization by Mass Spectrometry

描述(由申请人提供)：人体内的细胞通过降解细胞蛋白，并在细胞表面呈I型MHC相关蛋白的片段，向免疫系统传达其健康状况。受过适当教育的细胞毒性T淋巴细胞(CTL)(CD8+T细胞)与细胞表面的I类MHC分子结合，采样呈现的蛋白质片段(肽)，并杀死那些由于病毒、细菌和寄生虫感染、组织移植和细胞转化(癌症)而表达新肽的细胞。由于细胞信号通路的失调是癌症的标志之一，我们假设由这些通路产生的I类MHC磷酸肽应该是用于癌症免疫治疗的优秀候选者。在白血病、黑色素瘤和结直肠癌的初步研究中发现的I类MHC磷酸肽在多名健康献血者中引发预先存在的、中央记忆的、T细胞召回反应。一些白血病患者缺乏对磷酸肽抗原的中枢记忆回忆反应，并与临床结果相关。在同种异体干细胞移植后，这种反应会恢复。这些结果有力地表明，I类MHC磷酸肽是人类免疫监测的肿瘤靶点，因此很可能是癌症免疫治疗的候选对象。今年春天晚些时候，发现的三种磷酸肽将成为黑色素瘤I期临床试验的对象。建议进行更多的研究，以完成我们对黑色素瘤、白血病和结直肠癌的研究，并开始对食道癌、肝癌和卵巢癌的I类MHC磷酸肽进行表征。还将进行进一步的研究，以确定由处方药或环境制剂诱导并导致危及生命的自身免疫性疾病的I类或II类选择肽的改变的谱系。还提出了开发新的质谱学技术，该技术将在色谱时间尺度上促进完整的治疗性抗体和其他蛋白质在高飞摩尔水平上的接近完整的氨基酸序列覆盖。这将通过蛋白质化学衍生化、对现有仪器硬件的改变、增强仪器内离子-离子化学的性能以及对仪器控制软件的修改来实现。这种方法应该会加速针对多种疾病的治疗性抗体的开发，并有助于对包含同一蛋白质分子上的多个翻译后修饰的蛋白质和蛋白质复合体进行完整的表征。

DESCRIPTION (provided by applicant): 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 cellulr 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 phosphopeptides presented on esophageal, liver and ovarian cancer. Additional research will be conducted to identify the repertoire of altered class I or class II sel-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.