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Identification of novel cancer-specific T cell targets by dissection of successful tumour-infiltrating lymphocyte therapy

Rius Rafael, Cristina 2019. Identification of novel cancer-specific T cell targets by dissection of successful tumour-infiltrating lymphocyte therapy. PhD Thesis, Cardiff University.
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Background – T cell-based cancer immunotherapy has emerged as the biggest development in cancer treatment since radiotherapy and is capable of inducing complete, durable remissions in end-stage cancer patients. It is believed that the key effector cells are cytotoxic CD8+ T cells, which can detect anomalies in the proteome of cells that have undergone cancerous transformation, through the recognition of peptides presented at the cell surface bound to human leukocyte antigen (HLA) class I molecules. Recognition of peptide-HLA is controlled by the αβ T cell receptor (TCR). My work focussed on successful tumour infiltrating lymphocyte (TIL) therapy for the treatment of melanoma, which involves in vitro expansion of T cells from a tumour lesion to large numbers (>1010 cells) and reinfusion back into the patient’s blood. I had access to stored samples from a cohort of patients that underwent a complete durable remission following TIL therapy for stage IV melanoma. These samples included the TIL infusion product, an autologous melanoma cell line and peripheral blood mononuclear cells before treatment and after cancer clearance. The main focus of my thesis was to dissect the persistent tumour-reactive TCR repertoire in an HLA*02:01 melanoma patient successfully treated by TIL therapy and to identify the antigenic-specificity of key T cell clonotypes using a combination of High throughput sequencing (HTS), combinatorial peptide library (CPL) screening and CRISPR/Cas9 technology. Results – When I initiated my studies, it became apparent that the current “gold standard” for the identification of antigen-specific T cells, fluorochrome-conjugated peptide-HLA multimers (usually tetramers) failed to detect many fully functional T cells bearing low affinity TCRs. I therefore set out to optimise this technique so that it was capable of detecting all cancer-specific T cells within patient-derived samples. HTS of the TCRs that responded to the patient-autologous cancer line allowed me to identify cancer-specific TCRs in the TIL infusion product that persisted in patient blood after successful treatment. Disruption of the HLA A*0201 allele revealed that most of this response was operated through HLA A2. Some persistent clonotypes were shown to respond to a wide range of HLA A2+ cancer cell lines from a range of different tissue origins. This broad tumoricidal activity suggested that these T cell clones responded to unknown, common, shared antigens. Application of a bespoke CPL-based epitope discovery pipeline identified two new HLA A2-restricted epitopes. Further analysis of one persistent clonotype identified an important new property that allowed it to recognise most types of cancer cell via HLA A*02:01. Conclusions – I showed that peptide-HLA tetramers can fail to detect relevant functional T cell clonotypes and that this technology underestimates biologically relevant antigen-reactive T cell populations. Dissection of the cancer-specific T cell response in the TIL infusion product and blood following complete remission identified T cell clonotypes that responded to cancer cell lines from a range of different tissue origins, suggesting that these T cells respond to unknown shared antigens. I showed that a bespoke CPL-based ligand presentation platform could identify new broadly-expressed HLA A*02:01-restricted epitopes. TCRs with these specificities might protect from a wide range of cancer types and make exciting candidates for further therapeutic exploration.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Medicine
Date of First Compliant Deposit: 10 March 2020
Last Modified: 10 Mar 2020 10:17

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