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

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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.