Triple negative breast cancer (TNBC's) is a highly aggressive and invasive subtype of breast cancer, typically characterised by the lack of estrogen receptor (ER), progesterone receptor (PR) and Human epidermal growth factor receptor 2 (HER2) with an inexplicable partiality towards African women. The acute heterogenicity and complexity of TNBC tumours, together with a lack of well-defined molecular targets, complicates prognosis of the diseases resulting in patient reliance on traditional therapies, like chemotherapy, radiotherapy, and surgery, which are associated with elevated incidence of adverse effects and relapse. A major contributor to the heterogenicity of TNBCs is the tumour microenvironment which is composed of tumour infiltrating lymphocytes (TILs), tumour cells, healthy cells, and tumour vasculature. TILs have commonly been used as a prognostic marker and show robust predictive value for TNBC. In-depth analysis of the TIL composition within TNBC tumours may provide greatly beneficial information for the development of newer tumour microenvironment changing therapies and could assist doctors in understanding what therapies a particular patient maybe susceptible to. Thus, the diagnosis and therapy of this disease may greatly benefit from improved molecular profiling and patient stratification. Precision medicine seeks to provide such a solution, by dividing patients into subpopulations based on disease-specific profiles. The identification of new molecular targets would provide the basis for development of novel therapies. To this end, one of the major aims of this thesis was to develop a phage display based screening technique which could be utilised to isolate novel TNBC specific cancer antibodies. Once selected these antibodies could be used to generate TNBC specific therapies. Specific monoclonal antibodies (mAbs) and derivatives thereof, have already been established as a revolutionary tool for drug delivery to cancerous cells. Such antibodies have been conjugated to cytotoxic drugs to form antibody-drug conjugates, which may exhibit multiple advantages over their unconjugated counterparts, but their general use in clinical application has been restricted due to developmental deliberations. Historical conjugation strategies used for the generation of ADCs commonly resulted in heterogeneous mixtures of ADC species, with varying drug-to-antibody ratios resulting in unpredictable pharmacologic characteristics and safety profiles. In more recent time, self-labelling tags such as Snaptag have provided a means of developing homogenised recombinant immunotherapeutics. Snaptag is a modified version of a human DNA repair enzyme, O6 - alkylguanine-DNA-alkyltransferase (AGT) which naturally removes alkyl residues from damaged DNA. The enzyme reacts specifically with benzylguanine (BG) derivatives via irreversible transfer of alkyl groups to cysteine residues forming stable end products. In this thesis, Snaptag technology, together with other antibody discovery and manipulation tools was used to develop a methodology allowing for the generation of disease specific fusion proteins. Specifically, these fusion proteins consist of single-chain antibody fragments genetically fused to snaptag, allowing for the generation of recombinant ADCs that could be used as a drug delivery system carrying any BG-modified drug to a disease specific targets. In addition, snaptag interacts with BG in a 1:1 stoichiometry giving rise to homogenised combination products which when fused to a scFv provides a fail-safe target-specific therapeutic option. In addition to antibody conjugates, one of the most promising of all mAb based therapies currently used, are checkpoint inhibitors. In a balanced immune response, immune activation is counteracted with immunoregulatory pathways such as checkpoint inhibition. These negative regulatory pathways are necessary for maintaining tolerance and preventing hyperactivation, and are governed by cell surface, inhibitory receptors known as ‘'checkpoint inhibitors''. Blocking of checkpoint pathways during chronic infections and cancer has been shown to improve T-cell functions leading to reduced viral load and tumour burden. These findings have been translated into clinical application where checkpoint inhibitors, which are monoclonal antibodies targeting CTLA-4, PD1, PD-L1 or other inhibitory ligands, have been used to block these inhibitory interactions. The main intention of this research was to develop a methodology which could be used to generate snaptag based recombinant fusion proteins with potential diagnostic and therapeutic applications. Several snaptag based fusion proteins were developed using the recommended methodology these included fusion proteins targeting breast cancer specific antigen BCK1, checkpoint inhibitors PDL1, B7.1/CD80 (interacts with CTLA-4),and TIL characterising markers CD3, CD4, CD8, CD19 and CD20. In addition, to demonstrate the versatility and robustness of this methodology we sought to develop a snaptag based fusion protein not targeting breast cancer related antigens. Zika virus, an emerging infectious disease, currently lacking specific therapies was chosen for this purpose. An scFv derived from antibodies targeting the the Zika-DIII envelop protein, which is essential to the viral infection cycle was used in the snap fusion protein. The resulting ZIKA-DII-snap fusion protein demonstrated specific binding to zika virus membrane fractions. This research demonstrates the feasibility of using snaptag technology as a state-of-the-art conjugation strategy capable of bypassing the challenges previously associated with using antibodies as an effective delivery system for therapeutic drugs. By combining the applicability of snaptag technology with other antibody isolation and manipulation tools we were able to generate several functional snaptag based recombinant fusion proteins. Establishment of this methodology represents an important first step in generating medically necessary, pharmaceutically acceptable immunoconjugates that is instrumental in shifting general therapy towards a more personalized precision medicine approach.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/38132 |
| Date | 19 July 2023 |
| Creators | Nundalall, Trishana |
| Contributors | Barth, Stefan, Naran Krupa |
| Publisher | Faculty of Health Sciences, Department of Integrative Biomedical Sciences (IBMS) |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Doctoral Thesis, Doctoral, PhD |
| Format | application/pdf |
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