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

可充当功能性 T 细胞抗原的天然加工药物肽加合物的定义

• 批准号: MR/R009635/1
• 负责人: Dean Naisbitt
• 金额: $ 66.07万
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FR009635%2F1
• 关键词: Definition; naturally; processed; drug; peptide

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.

药物不良反应是一个主要的健康问题，也是新药开发的一个障碍。在英国，大约每16例住院患者中就有1例是由于某种形式的药物不良反应。最广为人知但最不为人所知的药物副作用之一是T淋巴细胞介导的超敏反应。就药物的化学成分和患者的生物学而言，这种反应是不可预测的。这项建议的目标是识别、表征和量化参与不良事件启动的化学信号(S)。为了实现这一目标，药物修饰的多肽将从表达在抗原提呈细胞(APC)表面的MHC分子中洗脱出来，并用质谱仪进行分析。然后将使用细胞培养方法来确定能够激活患者和健康捐赠者的T细胞的药物-肽加合物。这些数据将提供产生药物-多肽加合物结合四聚体的框架，以确定超敏和耐受患者PBMC中的T细胞数量和健康捐赠者的T细胞前体频率。尽管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，还适用于其他类别的药物，包括新药。我们最近利用质谱学来表征特定氨基酸残基上的药物-蛋白质结合情况与剂量和孵育时间的关系，并确定与T细胞激活相关的最低修饰水平。此外，初步研究已经表征了在MHC的背景下由APC展示的氟氯西林-多肽加合物。我们现在将使用这些方法来表征和量化MHC分子所展示的天然的、药物依赖的和药物修饰的免疫肽。洗脱肽的动力学分析和多肽展示中个体间变异性的评估将使我们能够识别这两个探针化合物的免疫优势药物修饰多肽。T细胞参与决定药物暴露是否会导致超敏反应的决策过程。因此，利用患者的PBMC和克隆的T细胞，我们将定义一种超敏表型，并鉴定参与T细胞激活的药物修饰的MHC结合肽。刺激性多肽的T细胞活性将与设计肽和经其他药物修饰的多肽进行比较，以探索结构特异性和肽骨架对T细胞反应的影响。对患者细胞的分析虽然重要，但无法提供有关初级T细胞反应的信息。因此，我们将利用我们的包含1000名健康捐赠者的PBMC的人类白细胞抗原基因分型细胞库来研究药物-多肽加合物特异性T细胞的来源。我们的逐步评估将包括：(1)筛选多个供体，以确定每个药物修饰多肽的反应者数量和每个个体中T细胞前光标频率；(2)评估供体反应最强的结构特异性，以及(3)细胞表型和功能的特征。药物修饰多肽与MHC和T细胞受体的相互作用将通过计算机模拟进行研究，以使我们更好地了解控制T细胞反应的机制。将通过X射线衍射法对与T细胞受体结合的MHC相关药物修饰多肽进行详细的结构分析。将针对一组药物修饰MHC结合多肽产生四聚体，以确定超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。将使用健康捐赠者的PBMC进行类似的分析，以探索T细胞光标前频率是否存在个体差异。最后，将使用组合四聚体染色来探索T细胞的交叉反应。

项目成果

The hydroxybutyric acid tolvaptan metabolite activates T-cells from healthy human donors and patients with drug-induced liver injury

羟基丁酸托伐普坦代谢物可激活健康人类捐赠者和药物性肝损伤患者的 T 细胞

• 发表时间: 2020
• 期刊: Hepatology
• 影响因子: 13.5
• 作者: Hammond S

Shared Clavulanate and Tazobactam Antigenic Determinants Activate T-Cells from Hypersensitive Patients.

• DOI: 10.1021/acs.chemrestox.2c00231
• 发表时间: 2022-11-21
• 期刊: CHEMICAL RESEARCH IN TOXICOLOGY
• 影响因子: 4.1
• 作者: Ariza, Adriana;Jaruthamsophon, Kanoot;Meng, Xiaoli;Labella, Marina;Adair, Kareena;Tailor, Arun;Sukasem, Chonlaphat;Whitaker, Paul;Peckham, Daniel;Pirmohamed, Munir;Torres, Maria Jose;Naisbitt, Dean John
• 通讯作者: Naisbitt, Dean John

Identification of flucloxacillin-modified hepatocellular proteins: implications in flucloxacillin-induced liver injury.

• DOI: 10.1093/toxsci/kfad015
• 发表时间: 2023-03-20
• 期刊: Toxicological sciences : an official journal of the Society of Toxicology

Detection of Hepatic Drug Metabolite-Specific T-Cell Responses Using a Human Hepatocyte, Immune Cell Coculture System.

• DOI: 10.1021/acs.chemrestox.2c00343
• 发表时间: 2023-03-20
• 期刊: CHEMICAL RESEARCH IN TOXICOLOGY
• 影响因子: 4.1
• 作者: Ali, Serat-E;Meng, Xiaoli;Kafu, Laila;Hammond, Sean;Zhao, Qing;Ogese, Monday;Sison-Young, Rowena;Jones, Robert;Chan, Benjamin;Livoti, Lucia;Sun, Yonghu;Sun, Lele;Liu, Hong;Topping, Anthony;Goldring, Christopher;Zhang, Furen;Naisbitt, Dean John
• 通讯作者: Naisbitt, Dean John

Activation of tolvaptan-responsive T-cell clones with the structurally-related mozavaptan.

使用结构相关的莫扎瓦普坦激活托伐普坦反应性 T 细胞克隆。

• DOI: 10.1016/j.toxlet.2022.11.017
• 发表时间: 2023
• 期刊: Toxicology letters
• 影响因子: 3.5
• 作者: Hammond S