Definition of the naturally-processed drug-peptide adducts that can act as functional T-cell antigens

药物不良反应是一个主要的健康问题，也是开发新药的障碍。在英国，大约有1 / 16的住院患者是由于某种形式的药物不良反应。其中一个最著名但最不为人所知的药物副作用是t淋巴细胞介导的过敏反应。就药物的化学性质和病人的生物学特性而言，这种反应是不可预测的。本提案的目的是识别、表征和量化与不良事件起始有关的化学信号。为了实现这一目标，药物修饰肽将从抗原提呈细胞（APC）表面表达的MHC分子中洗脱出来，并通过质谱分析。然后，细胞培养方法将用于确定激活来自患者和健康供体的t细胞的药物肽加合物。这些数据将为生成药物-肽加合物结合四聚体提供框架，以确定过敏和耐受PBMC患者的t细胞数量和健康供者的t细胞前体频率。虽然我们将重点研究两种β -内酰胺类抗生素，哌拉西林和氟氯西林，但研究结果不仅适用于其他抗生素，也适用于其他类别的药物，包括新药。我们最近利用质谱法来表征与剂量和孵育时间相关的特定氨基酸残基的药物-蛋白质偶联谱，并确定与t细胞激活相关的最小修饰水平。此外，初步研究已经表征了APC在MHC背景下显示的氟氯西林肽加合物。我们现在将使用这些方法来表征和量化天然的、药物依赖的和药物修饰的MHC分子所显示的免疫肽穹窿。洗脱肽的动力学分析和肽展示的个体间变异性评估将使我们能够确定两种探针化合物的免疫优势药物修饰肽。t细胞参与决定药物暴露是否会导致过敏反应的决策过程。因此，利用PBMC和克隆的患者t细胞，我们将定义一种超敏表型，并鉴定参与t细胞活化的药物修饰的mhc结合肽。刺激肽的t细胞活性将与设计肽和其他药物修饰的肽进行比较，以探索结构特异性和肽主链对t细胞反应的影响。对患者细胞的分析虽然很重要，但却不能提供有关原代t细胞反应的信息。因此，我们将利用我们的HLA基因型细胞库，其中包含来自1000名健康供体的PBMC，来研究药物肽加合物特异性t细胞的来源。我们的逐步评估将包括(1)筛选多个供体，以确定每种药物修饰肽的应答数量和每个个体的t细胞前游标频率；(2)评估具有最强反应的供体的结构特异性；(3)细胞表型和功能的表征。药物修饰肽与MHC和T细胞受体的相互作用将通过计算机模拟进行研究，以使我们更好地理解控制T细胞反应的机制。MHC相关药物修饰肽与t细胞受体复合物的详细结构分析将通过x射线衍射进行。将针对一组药物修饰的mhc结合肽生成四聚体，以确定过敏和耐受患者中抗原特异性t细胞的数量，并使用流式细胞术进行详细的表型分析。将对健康供体的PBMC进行类似的分析，以探索t细胞前游标频率是否存在个体差异。最后，组合四聚体染色将用于探索t细胞的交叉反应性。

Adverse drug reactions are a major health concern and an impediment to the development of new medicines. Approximately 1 in 16 hospital admissions in the UK are due to some form of adverse drug reaction. One of the best known, but least understood drug side-effects is T-lymphocyte-mediated hypersensitivity. Such reactions are unpredictable with respect to the chemistry of the drug and the biology of the patient. The objective of this proposal is to identify, characterize and quantify the chemical signal(s) involved in the initiation of the adverse event. To fulfil this objective, drug-modified peptides will be eluted from MHC molecules expressed on the surface of antigen presenting cells (APC) and analysed by mass spectrometry. Cell culture methods will then be used to define the drug-peptide adducts that activate T-cells from patients and healthy donors. These data will provide the framework to generate drug-peptide adduct binding tetramers to determine the number of T-cells in hypersensitive and tolerant patient PBMC and the T-cell precursor frequency in healthy donors. Although we will focus our research on two beta-lactam antibiotics, piperacillin and flucloxacillin, the findings will be applicable not only to other antibiotics, but also other classes of drugs including novel medicines.We have recently utilized mass spectrometry to characterize the profile of drug-protein conjugation at specific amino acid residues with respect to dose and incubation time and to define the minimum level of modification associated with the activation of T-cells. Furthermore, preliminary studies have characterized flucloxacillin-peptide adducts displayed by APC in the context of MHC. We will now use these methods to characterize and quantify the natural, drug-dependent and drug-modified immunopeptidome displayed by MHC molecules. Kinetic analysis of eluted peptides and assessment of inter-individual variability in peptide display will allow us to identify immunodominant drug-modified peptides for the two probe compounds.T-cells are involved in the decision process that determines whether drug exposure will lead to a hypersensitivity reaction. Thus, using PBMC and cloned T-cells from patients, we will define a hypersensitivity phenotype and identify the drug-modified MHC-binding peptides involved in T-cell activation. T-cell activity of stimulatory peptides will be compared with designer peptides and peptides modified with other drugs to explore structural specificity and the influence of the peptide backbone on the T-cell response.Analysis of patient cells, although important, provides no information about the primary T-cell response. Thus, we will utilize our HLA genotyped cell bank containing PBMC from 1000 healthy donors to study the origin of drug-peptide adduct-specific T-cells. Our stepwise assessment will involve (1) screening multiple donors to determine the number of responders to each drug-modified peptide and the T-cell pre-cursor frequency in each individual; (2) assessment of structural specificity with donors displaying the strongest responses, and (3) characterization of cellular phenotype and function.The interaction of drug-modified peptides with MHC and T-cell receptors will be investigated by in silico modelling to allow us to better understand the mechanisms that govern T cell responses. Detailed structural analysis of MHC associated drug-modified peptides in complex with T-cell receptors will be performed by X-ray diffraction.Tetramers will be generated against a panel of drug-modified MHC-binding peptides to ascertain the number of antigen-specific T-cells in hypersensitive and tolerant patients and to derive detailed phenotypic analysis using flow cytometry. A similar analysis will be conducted using PBMC from healthy donors to explore whether individual differences in T-cell pre-cursor frequency exist. Finally, combinatorial tetramer staining will be used to explore T-cell cross-reactivity.