Agliardi, Giulia;
(2017)
Development of a Chimeric Antigen Receptor (CAR) - based T cell therapy for glioblastoma.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
High grade gliomas are aggressive brain tumours for which treatment is highly challenging due to the location within the central nervous system (CNS), which may reduce access of cytotoxic chemotherapy, and their infiltrative growth, which precludes complete surgical resection. Current treatment includes surgical removal – wherever possible - followed by radiotherapy and chemotherapy. However, recurrence is common, resulting in a survival of only 12 to 15 months after diagnosis. This highlights the need for new therapies. Chimeric antigen receptors (CARs) are synthetic molecules which combine the specificity of an antibody to the signalling domains of a T cell receptor (TCR), allowing T cells to directly recognise tumour antigens with no need for co-stimulation. CAR-T cells have shown promising responses in the treatment of haematological malignancies, inducing complete and durable responses in patients with chemo-refractory disease treated with CD19-redirected T cells. This therapeutic approach may be highly suitable for high grade gliomas as T cells have the ability to track to distant tumour sites. However, translation of this technology to solid tumours is proving more difficult, due to several challenges, including: requirement for an effective infiltration of CAR-T cells within the tumour and the immunosuppressive environment provided by solid malignancies. In this work, we developed an immunocompetent animal model of glioma, to study kinetics of migration and infiltration of CAR-T cells and the interplay between CAR-T cells, the tumour and the endogenous immune system to inform the design of T cell immunotherapy for this brain tumours. The tumour specific variant III of the epidermal growth factor receptor (EGFRvIII) – a mutation found in 30% of glioblastomas – was used as model antigen. A murine CAR was constructed based on the single chain fragment variant (ScFv) of EGFRvIII-specific antibody MR1.1 linked with a CD8 stalk to CD28-CD3ζ activation domains. A murine marker gene (truncated CD34) was co-expressed to allow for ex vivo analysis as well as firefly luciferase for in vivo tracking of CAR T-cells. The mouse glioma cell line GL261 was modified to express the mouse version of EGFRvIII and used to establish orthotopic tumours. After validation of function and specificity in vitro, efficacy of CAR-T cells was tested in vivo. Both bioluminescence imaging (BLI) and flow cytometry demonstrated that CAR T cells accumulated within the tumour in an antigen-dependent manner. MRI demonstrated that CAR T cells delayed tumour growth and increased survival. However, tumours were not consistently eradicated. Both immunohistochemistry and BLI indicated lack of long term persistence of T cells within the tumour. Analysis of tumour infiltrating lymphocytes (TILs) phenotype suggested that decreased functionality of CAR-T cells could be a result of their exhaustion in situ. We hypothesised that additional strategies were required to improve efficacy and persistence of CAR-T cells. We postulated that CAR-T cell fitness may be prolonged by: - Incorporation of 41BB as additional co-stimulatory domain in the CAR to provide a pro-survival signal. - Combination therapy with PD1 blockade to overcome T cell exhaustion (both on CAR and endogenous T cells) in situ. While the employment of third-generation CAR did not significantly improve survival and showed increased toxicity, combination therapy of CAR-T cells and PD-1 blockade promoted complete clearance of tumours resulting in long term survival. Immunohistochemistry and flow cytometry analysis suggested that combination therapy may increase persistence of CAR-T cells, leading to a more rapid and consistent tumour eradication compared to CAR-T cell administration alone. However, data presented here did not demonstrate a synergistic effect of CAR-T cell therapy and PD1 blockade, as an effect of PD1 blockade alone was also observed. Therefore, additional experiments are required to examine this further.
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