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

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.

Background: Drug hypersensitivity reactions cannot be predicted and show no simple dose-response relationship. Recently, we have demonstrated that beta-lactam-protein conjugates can function as antigens for T-cells and that the same conjugates are formed in patients. We have also shown that is possible to characterize natural drug-modified MHC binding peptides. Despite this, the peptide repertoire displayed by MHC molecules on antigen presenting cells exposed to drugs, the structure of the drug-modified peptide T-cell receptor binding interaction and the drug-modified peptides that activate T-cells have not been defined.
Objectives: We propose to conduct a kinetic analysis of the drug-modified and unmodified immunopeptidome. Moreover, we will synthesize drug-modified peptides to identify the antigenic determinants that activate T-cells from hypersensitive patients and healthy donors. Structural assessment of MHC associated drug-modified peptides in complex with T-cell receptors will be performed, while tetramers will be used to ascertain antigen-specific T-cell numbers in hypersensitive and tolerant patients and healthy donors.
Methods: We will characterize drug-modified MHC binding peptides and the kinetics of MHC peptide display using mass spectrometry and directly relate the chemistry of antigen formation to immune function using PBMC from hypersensitive patients and healthy donors. Immune responses will be measured using a battery of established methods and through the production of tetramers. The GOLD programme will be used for in silico modelling, while X-ray diffraction will define the structure of drug-modified peptides bound to MHC and T-cell receptors.
Impact: A better understanding of drug hypersensitivity will improve our ability to diagnose, predict and prevent such reactions. Synthesis of drug-modified peptides as reagents will allow the development of more evidence-based culture systems and thereby improve drug safety for the good of public health.

We propose a new approach to understand the chemical basis of drug hypersensitivity. Cutting edge technologies will be used to identify and characterize naturally processed drug-modified peptides to ascertain those structures responsible for eliciting life-threatening T-cell reactions. The data generated will provide a rationale-based approach for developing robust in vitro assays using drug-modified peptides for improved hypersensitivity diagnosis in patients and prediction of immunogenicity during drug development. The project will also provide us with basic insights into immunology which may be applicable to other areas, for example chemical allergy, cancer therapy and autoimmunity. The need to study mechanisms of drug hypersensitivity for the development of improved diagnostic tests is the subject of a recent review by an EAACI taskforce (Mayorga et al. Allergy. 2016;71:1103-34).

Who are the beneficiaries of the research?
1. Pharma: Drug hypersensitivity reactions are usually not detected until a drug is administered to a large population, such as phase III clinical trials, or the market. As well as a risk to human safety, the withdrawal of drugs at such a late stage is of great cost to Pharma, and also prolongs the time for the patient until a safer drug is developed. The cost of discovering and developing a drug is estimated to be about $900 million, and takes approximately 13 years. Through an interdisciplinary approach that relates donor genotype and phenotype to the chemical characteristics of the drug antigen we will define the fundamental pathways underpinning the activation of drug-specific T-cells. The scientific knowledge that we will generate will provide important insights into the development of tools/assays for use in pre-clinical screens to identify drug hazard; thus, reducing late-stage drug withdrawals and the cost and time to bring safer drugs to the market (timeline 3-5 years).

2. Patients with drug hypersensitivity and health service providers: The high prevalence of beta-lactam hypersensitivity reactions limits antibiotic choice and results in patients being excluded from treatment or receiving suboptimal therapy. Existing in vitro diagnostic assays and skin testing lack sensitivity. This results in patients being labelled as hypersensitive according to clinical features alone and told to avoid subsequent exposure to beta-lactam antibiotics. A number of these patients will have been mis-diagnosed, while many more will be able to safely tolerate alternative drugs. New insights into the drug-modified peptides that activate T-cells will identify novel mechanism-based reagents for use in diagnostic tests and as a consequence, it will be possible to de-label certain hypersensitive patients and define the culprit drug in others.

Antibiotic hypersensitivity has major implications for health service providers. Patients who have suffered from hypersensitivity reactions are often older and sicker and require more frequent treatment. Alternative antibiotics can cost more per treatment course than first line drugs. Improved diagnosis of reactions will allow clinicians to prescribe antibiotics more appropriately (timeline 5 years).

3. Diagnostics sector: Our findings will pave the way for the development of sensitive and specific diagnostic tests that increase drug safety by stratification of drug use. The concept of such tests would be attractive to diagnostic companies for future development and commercialisation (timeline 5 years).

4. Drug regulators: Scientific outputs from the project will better inform drug regulators and will thereby contribute towards better risk-benefit assessments for the use of drugs (timeline 5 years).

5. Drug safety scientists: The project will bring new researchers with fresh insights into the field of Drug Safety Science. They will learn to handle clinical samples and receive training in drug bioanalysis and immunology.