title: DEVELOPMENT OF IN VITRO CELL-BASED SYSTEMS TO INVESTIGATE DRUG IMMUNOGENICITY
Creators: name: Ali, Serat-E
Publisher: name: University of Liverpool Repository
Year: 2022

description: Typically, drugs are too small to elicit an immune response and therefore the occurrence of drug-mediated reactions is extremely rare. Characteristically these reactions are inherently difficult to predict from only the pharmacology of a drug or from the genotype and phenotype of the patient. Current models such as the lymphocyte transformation cells and drug challenge tests fall short of providing a panacea due to their invasive nature and inaccurate predictive capabilities. The ability to model hypersensitivity reactions in vitro would therefore prove useful in the clinic and during design, preventing morbidity and mortality. An ideal model would mimic the immune microenvironment through incorporating drug, patient, and disease specific factors. In the studies described in this thesis, we aimed to develop tools for future hypersensitivity modelling, encompassing drug, patient and disease specific factors alongside the immune microenvironment. We focused on models which may help predict chemically reactive metabolite formation resulting in T-cell activation, incorporating drug and patient factors. We utilised drug-metabolite specific T-cell clones, donor specific antigen presenting cells and a metabolising system to demonstrate chemically reactive metabolite formation and subsequent T-cell activation. Dapsone-nitroso specific T-cell clones demonstrated strong proliferative responses when cultured in situ dapsone and a metabolising system. Furthermore, through mass spectrometry analysis, we demonstrate the formation of multiple chemically reactive metabolites within the cell culture medium. We next investigated how the tissue microenvironment is related to flucloxacillin accumulation and novel adduct formation in flucloxacillin-mediated drug induced liver injury. Utilising the HepaRG cell line and primary human hepatocytes, evidence suggests that MRP2 and P-gp expression and function may play a role in flucloxacillin accumulation in liver-like cells. Furthermore, utilising membrane transporter inhibitors, we demonstrated how disruption of transporters increases the formation of flucloxacillin modified proteins and peptides for possible T-cell presentation and activation, contributing to the DILI mechanism. We next aimed to incorporate the HLA-predisposition of flucloxacillin in drug induced liver injury alongside the target tissue, creating an autologous T-cell mediated cytotoxicity model. Through use of donor specific induced pluripotent stem cells, T-cell clones and antigen presenting cells, we were able to demonstrate flucloxacillin-specific T-cell mediated killing of HLA-B*57:01 hepatocyte-like cells. Finally, utilising traditional and well-defined T-cell models, we demonstrated how the use of a conventionally safe therapeutic moiety, green tea, may result in a hypersensivity reaction, given the right patient and disease factors. T-cell isolation from HLA-B*35:01 donors demonstrated a strong dose-proliferation relationship upon exposure to epigallocatechin gallate and green tea extract.