E-Thesis 316 views
Characterising novel Antibody-Drug Conjugate targets for ovarian cancer: Implications for patient stratification and precision medicine / ALICE LUTHER
Swansea University Author: ALICE LUTHER
DOI (Published version): 10.23889/SUthesis.58415
Abstract
Ovarian cancer (OC) is the 6th most common cancer in females and accounts for 5% of all female cancer deaths in the UK. OC presents with ambiguous symptoms often leading to a late diagnosis. Only 42.6% of women diagnosed with OC will survive for five years or more with many not responding to current...
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Swansea
2021
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| Institution: | Swansea University |
| Degree level: | Doctoral |
| Degree name: | Ph.D |
| Supervisor: | Gonzalez, Deyarina. ; Del Sol, Ricardo ; Conlan, Robert S. |
| URI: | https://cronfa.swan.ac.uk/Record/cronfa58415 |
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| Abstract: |
Ovarian cancer (OC) is the 6th most common cancer in females and accounts for 5% of all female cancer deaths in the UK. OC presents with ambiguous symptoms often leading to a late diagnosis. Only 42.6% of women diagnosed with OC will survive for five years or more with many not responding to current treatments. Platinum-based drugs are the most widely used chemotherapy drugs in ovarian carcinoma, but their efficacy is not satisfactory. Bevacizumab and PARP inhibitors are the only targeted therapies currently approved for the treatment of this disease. Still, most patients will still recur and unfortunately succumb to this disease, thus new and more effective treatments and diagnosis methods are urgently needed. Antibody-Drug Conjugates (ADCs) are a group of targeted therapeutic molecules that have proved efficacious as therapeutic options for the treatment of various cancers such as Acute Myeloid Leukaemia and Hodgkin Lymphoma. Currently there is not an ADC approved for the treatment of ovarian cancer. ADCs couple the targeting activity of a monoclonal antibody with the cytotoxic activity of a potent small molecule drug to selectively destroy malignant cells while leaving the healthy cells unaffected. The discovery of novel ADC targets for the treatment of OC is crucial in combatting the poor prognosis associated with late-stage ovarian cancer. The late-stage diagnosis of OC is significantly associated with a reduced chance of survival. Confirmation of OC diagnosis is achieved through histological examination of biopsy tissue taken at surgery. This current diagnosis process is not appropriate for the high-throughput screening needed for the early detection of OC that could be offered if minimally invasive high-throughput liquid biopsy diagnostic tests are developed for OC. Objectives•To identify and characterise transmembrane proteins as potential ADC targets•To confirm the genetic stability of the antibody epitope DNA coding sequence in the Receptor for Advanced Glycation End products (RAGE) and the lead candidate ADC target•To explore whether soluble RAGE (sRAGE) levels could serve as OC biomarker with diagnostic value using an Enzyme Linked Immunosorbent Assay (ELISA) liquid biopsy pipelineMethodology This study used western blots, immuno-fluorescent microscopy, internalisation assays by pH reactive dye, Illumina Next Generation Sequencing and Enzyme-Linked Immunosorbent assay. 2D cultured cells and protein were derived from primary patient biopsies. Eight immortalised ovarian cancer cell lines were used (See section 2.2.1.1). Results Immunofluorescent microscopy, immunoblotting and internalisation assay data in both ovarian cancer cell lines and cells derived from primary ovarian cancer patient biopsies revealed Tetraspanin 6 (TSPAN6) as the lead candidate target for novel ADC development. Not a lot is known about TSPAN6 function however it has been reported to mediate signal transduction events that play a role in the regulation of cell development, activation, growth, and motility. Three targets showed localisation to the cell membrane in ovarian cancer cells. Two targets (PCSK4 and TSPAN6), plus RAGE, showed antibody-internalisation capacity and translocation to the lysosomal compartment in cancer cells demonstrating their suitability as ADC targets. RAGE and TSPAN6 showed genetic stability by Illumina Next Generation Sequencing deep amplicon variant analysis. A novel TSPAN6 60 kDa immunoreactive protein band was identified in protein derived from a 3D primary tissue source and it significantly stratified OC from non-cancer patients. The ELISA technique was successful in detecting sRAGE in patient serum, however soluble serum RAGE levels did not exhibit a potential diagnostic value for OC disease in the clinical OC specimens analysed. Conclusions This thesis identified TSPAN6 as a novel ADC target for OC. In addition, both TSPAN6 and RAGE epitopes are highly conserved in humans, suggesting their promising therapeutic value for the development of immunotherapies. Nevertheless, levels of soluble serum RAGE are not indicators of an OC diagnosis. |
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| Item Description: |
A selection of third party content is redacted or is partially redacted from this thesis due to copyright restrictions. |
| Keywords: |
ADC, novel targets, discovery pipeline, ovarian cancer, TSPAN6, precision medicine, western blot, immunofluorescent microscopy, ELISA, NGS |
| College: |
Faculty of Medicine, Health and Life Sciences |