Medical Biology - Theses

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Start date: 2000 × Subject: Brain cancer ×

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    Characterisation of dual-specific Chimeric Antigen Receptor T cells against heterogeneous tumours
    Hughes-Parry, Hannah Emily ( 2023-06)
    The heterogeneity of solid tumours is a significant obstacle to the response and long-term remission of patient malignancies following Chimeric Antigen Receptor (CAR) T cell immunotherapy in the clinic. While long-term remission has been achieved against haematological cancers, relapses have frequently occurred several months post-treatment due to antigen escape, and solid tumour responses have been less effective. Therefore, to improve solid tumour elimination and prevent relapse, CAR T cell immunotherapy may be improved by the targeting of multiple tumour-associated antigens through dual-specific CAR T cells, in which T cells are engineered to express CARs against multiple antigens. Existing studies have observed significant improvements over single-specific CAR T cells; however, few studies have interrogated the underlying biology in immune competent systems. In this thesis, I explored whether dual-targeting CAR T cells targeting the HER2 and EGFRvIII tumour antigens were able to effectively clear heterogeneous tumours both in vitro and in vivo. I assessed their cytotoxic function and cytokine secretion against different heterogeneous tumour targets in vitro. I found that dual-specific CAR T cells exhibit enhanced killing of heterogeneous tumour cells, but not elevated levels of cytokine secretion or exhaustion markers compared to single-specific and pooled single-specific CAR T cells. This enhanced ability for multi-antigen targeting T cells to eliminate heterogeneous tumours allows for more complete clearance of the entire tumour cell population and may subsequently mitigate opportunities for antigen escape. To explore the utility of multitargeting tumour antigens in vivo, we used CRISPR technology to generate an immunocompetent mouse model (RHEO), tolerant to human HER2, EGFRvIII and OVA, to evaluate dual-targeted CAR T cell immunotherapy approaches. I demonstrate that administration of dual CAR T cells results in improved survival in vivo in RHEO mice using a heterogeneous intracranial tumour model, and a combination therapy, by combining dual CAR T cells with anti-CD137 agonist, results in complete intracranial tumour clearance in all mice. These results highlight the importance of evaluating CAR T cell efficacy in an immunocompetent mouse model. While targeting the entirety of a heterogeneous tumour with multi-targeting CAR T cells is critical for tumour elimination, combining CAR T cell immunotherapy with other immune modulatory agents may be necessary to achieve complete tumour clearance.
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    Engineering Chimeric Antigen Receptor T Cell Therapy for Glioblastoma
    Abbott, Rebecca Caitlyn ( 2022)
    Glioblastoma is a highly aggressive form of brain malignancy which primarily affects adults, but cases do also arise in children. The current standard of care for glioblastoma; the Stupp protocol, consists of debulking surgery, chemotherapy and radiotherapy. However, the treatment is highly unlikely to be curative and tumours commonly relapse. The current lack of effective treatments for glioblastoma highlights the need for the development of precision therapies. The immune system possesses a natural capability to protect the body and the anti-tumour functionality of T cells has been harnessed in the development of novel treatments. Chimeric Antigen Receptor (CAR) T cells express a synthetic cell surface receptor specific for a tumour-expressed antigen. These cells can then exert cytotoxic and cytokine-producing functions directed towards tumour cells. In a subset of glioblastoma patients, the Epidermal Growth Factor Receptor (EGFR) protein is truncated, generating an exquisitely tumour-specific mutation (EGFRvIII) to target with precision-based medicines. In this thesis, a novel CAR targeting this protein; GCT02, was characterised in primary murine and human T cells. The prediction of the binding epitope indicated GCT02 to bind to amino acids in a shared region between the EGFR and EGFRvIII proteins. Despite this, the CAR demonstrated functional specificity to cells expressing EGFRvIII in both the murine and human systems in vitro. In vivo, a single infusion of the GCT02 CAR T cells mediated the clearance of implanted orthotopic glioblastoma tumours within one month post T cell infusion. The CAR T cells were unreactive to primary human EGFR+ keratinocytes and astrocytes, suggesting a favourable safety profile. Unfortunately, the clinical evaluation of EGFRvIII specific CAR T cells has led to disappointing anti-tumour efficacy. Consequently, this thesis also investigated the re-utilisation of the GCT02 binding domain into a synthetic Notch receptor, employing logic gating technology. The system is designed to restrict the induced secretion of anti-tumour factors to within the tumour site, limiting systemic toxicity. Unfortunately, whilst the constructs could be generated and highly expressed in the Jurkat human T cell line, the circuit failed to activate in the presence of an EGFRvIII stimulus. The work in this thesis has directly contributed to the establishment of the CAR T cell development pipeline in the Jenkins Laboratory whilst characterising a novel, high affinity antigen binding domain which shows efficacy in a CAR format and may also have further clinical applications given the favourable safety profile.