Development of a stepwise screening approach to assess the intrinsic immunogenicity of drugs and chemicals

药物不良反应是新药开发的严重障碍。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。最著名的，但最不为人所知的药物副作用之一是过敏。就药物的化学性质和人体生物学而言，这种反应是不可预测的。因此，临床前动物模型无法预测一种新化合物是否会在人体中引起免疫反应。因此，我们设想了一个多学科项目，使用来自健康供体的细胞来定义药物和免疫受体之间的化学界面，以及药物受体结合相互作用发展成异常免疫反应需要克服的检查点。这一机制免疫学将为检测工具箱提供框架，可以逻辑方式用于评估药物特异性免疫反应的质量和强度。分层分析将产生一个筛选平台，在药物开发的早期和后期阶段评估新型候选药物的内在免疫原性。为了使这个项目可行，我们已经建立了一个HLA基因型细胞库，其中包含来自1000名健康供体的血液单个核细胞，并建立了研究药物和药物代谢物特异性t淋巴细胞反应起源所需的培养条件。这为在基础细胞、分子和化学水平上研究任何已知药物HLA相关性提供了生物学资源。我们最初的研究将集中在一组与高过敏反应发生率相关的化合物（即训练化合物）上，以产生抗原特异性t细胞反应，并确定HLA等位基因在抗原呈递中的作用。这种已建立的方案耗时，并且是为利用单个drug-naïve供体的细胞进行基于实验室的少数变量调查而设计的。为了形成一个合适的药物筛选平台，我们将专注于小型化，以评估(1)同时评估多个hla型个体，(2)在单个平板格式中使用来自一个个体的细胞进行多次实验。已建立的药物免疫原性筛选平台（1级试验）将转移给我们的工业合作伙伴，盲法试验化合物将在两个地点进行评估，以确定试验的敏感性和特异性。细胞系统中的药物配置、免疫参数和诱导反应所需的人为因素的基本知识尚未完全定义，因此尚未应用于相关的测试系统。因此，我们将进行机制免疫学研究，以阐明(1)药物与特异性HLA等位基因的结合是否会导致naïve和/或记忆t细胞的激活，(2)调节药物特异性t细胞激活的参数，(3)组织细胞中形成的药物蛋白加合物，以及药物抗原在组织细胞上表达的MHC分子上的展示是否会导致t细胞激活。我们已经克服了开发自体组织/免疫细胞共培养系统的主要挑战，通过生成ips衍生的肝细胞和角化细胞系以及来自同一hla型健康供体的抗原特异性t细胞。这些实验将构成二级分析的基础，以探索药物特异性t细胞反应的性质以及调节和组织因子。3级分析包括从1级和2级分析中克隆药物和药物代谢物特异性t细胞，以确定细胞表型和功能，并评估HLA等位基因限制、交叉反应性和t细胞活化机制。该项目的最终结果是推荐一系列可用于筛选新化合物潜在免疫原性的检测方法。与我们的工业合作伙伴一起工作，我们有一个筛选分析在现实生活中初始应用的途径。

Adverse drug reactions are a serious 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 hypersensitivity. Such reactions are unpredictable with respect to the chemistry of the drug and the human biology. For this reason, pre-clinical animal models fail to predict whether a novel compound will induce an immune response in humans. Thus, we have conceived a multi-disciplinary project using cells from healthy donors to define the chemical interface between drugs and immune receptors, and the checkpoints that need to be overcome for the drug receptor binding interaction to develop into an aberrant immune response. This mechanistic immunology will provide the framework for a toolbox of assays that can be used in a logical fashion to assess the quality and intensity of drug-specific immune responses. Stratification of assays will result in a screening platform to assess intrinsic immunogenicity of novel drug candidates at early and later phases of drug development.For this project to be feasible, we have established an HLA genotyped cell bank containing blood mononuclear cells from 1000 healthy donors and the culture conditions needed to study the origin of drug- and drug metabolite-specific T-lymphocyte responses. This provides the biological resource to investigate any known drug HLA association in the laboratory at the fundamental cellular, molecular and chemical level. Our initial research will focus on a panel of compounds associated with a high incidence of hypersensitivity reactions (i.e., training compounds) to generate antigen-specific T-cell responses and to define the role of HLA alleles in antigen presentation. This established protocol is time-consuming and designed for laboratory-based investigation of few variables utilizing cells from a single drug-naïve donor. In order to form a suitable platform for the screening of drugs we will focus on miniaturization to assess (1) multiple HLA-typed individuals simultaneously and (2) multiple experiments with cells from one individual within a single plate format. Established drug immunogenicity screening platforms (level 1 assays) will be transferred to our industrial partner and blinded test compounds will be assessed at both sites to define assay sensitivity and specificity.The essential knowledge of drug disposition in cell systems, immune parameters and human factors required to induce a response have not been fully defined and have therefore not been applied to a relevant test system. Thus, we will conduct mechanistic immunological investigations to elucidate (1) whether drug binding to specific HLA alleles results in the activation of naïve and/or memory T-cells, (2) the parameters that regulate activation of drug-specific T-cells (3) the drug protein adducts formed in tissue cells and whether the display of drug antigens on MHC molecules expressed on tissue cells results in T-cell activation. We have already overcome the major challenge of developing autologous tissue/immune cell co-culture systems through the generation of iPS-derived hepatocyte and keratinocyte lines and antigen-specific T-cells from the same HLA-typed healthy donors. These experiments will form the basis for level 2 assays to explore the nature of drug-specific T-cell responses and regulatory and tissue factors.Level 3 assays involve the cloning of drug- and drug metabolite-specific T-cells from level 1 and 2 assays to define cellular phenotype and functionality and to assess HLA allele restriction, cross reactivity and mechanisms of T-cell activation.The ultimate outcome of the project is to recommend a series of assays that can be used to screen for potential immunogenicity of new compounds. Working alongside our industrial partner we have a pathway for initial application of the screening assays in a real-life setting.