Development of a new therapeutic regime for the treatment of glioblastoma multiforme (GBM)

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Pearson, J.R.D., 2019. Development of a new therapeutic regime for the treatment of glioblastoma multiforme (GBM). PhD, Nottingham Trent University.

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Abstract

Glioblastoma multiforme is the most frequently occurring primary brain tumour and it carries a dismal prognosis. Despite surgical intervention and aggressive chemo-radiotherapy the median survival of patients is just under 15 months. Very little progress has been made with regards to improving patient survival therefore novel therapeutic interventions are required.

Vaccination offers an attractive option for the therapeutic treatment of GBM, with activated T-cells previously being shown to access tumours located within the brain resulting in improved survival in pre-clinical murine models of GBM. Several vaccine platforms have been developed and tested in GBM patients, however many of these therapies fail during clinical trials and as of 2019 no immunotherapeutic treatments have been approved for use in the GBM setting. One major obstacle to overcome when using the immune system to treat GBMs is the highly immunosuppressive phenotype these cancers have. GBMs frequently express immunosuppressive checkpoints that prevent anti-tumour T-cell activity. Immune checkpoint blockade has recently gained approval for the treatment of malignant melanoma, lung cancer, head and neck cancer, lymphoma, bladder cancer and kidney cancer. The utilisation of immune checkpoint blockade as a monotherapy improves survival in small subsets of patients however it is not a completely curative treatment modality. Therefore it is of great interest to see if combining immune checkpoint blockade with vaccine therapy can boost anti-tumour immunity by stimulating T-cells and preventing the inhibitory signals from tumour cells that prevent T-cell killing.

Examination of GBM tumour tissues and cell lines revealed that a large proportion of GBMs express the immunogenic antigens Tyrosinase-related protein-2 (TRP-2) and Wilms’ tumour 1 (WT-1). It was also revealed that these tumours expressed several immunosuppressive proteins with PD-L1, HLA E and HLA-G expression being observed in tissues and cell lines studied. It was also revealed that when GBM cell lines were treated with the immune stimulating cytokine IFNγ they up-regulated the immunosuppressive proteins PD-L1 and IDO.

The ImmunoBody® DNA plasmid vaccination encodes an IgG antibody molecule that acts as a carrier protein for the peptide targets of interest, these peptides are engrafted into the complementarity determining regions of the antibody. This method of vaccination generates a strong-immune response via direct and cross-presentation. Pre-clinical testing using the humanised HHDII/DR1 C57BL/6 mouse model revealed that a HLA-A2 specific TRP-2 and a HLA-A2 specific WT-1 directed ImmunoBody® vaccine generated a strong peptide specific immune response. When both vaccinations were given simultaneously this strong TRP-2 and WT-1 directed immune response was equivalent to when each vaccine was given alone meaning that epitope dominance is not a factor when targeting these two antigens.

Using the HHDII/DR1 humanised mouse the effects of this dual ImmunoBody® vaccination regime was tested both prophylactically and therapeutically. In these proof of concept experiments the B16 HHDII/DR1 Luc2 cell line was utilised. This cell line expresses both TRP-2 and WT-1 antigens and it has the chimeric HLA-A2 HHDII/DR1 MHC molecule meaning it presents HLA-A2 specific peptides. When the dual ImmunoBody® vaccination regime was used prophylactically it significantly improved the survival of mice intracranially implanted with B16 HHDII/DR1 Luc2 cells compared to sham vaccinated mice. In the therapeutic setting the addition of an anti-PD-1 blocking antibody to the dual vaccination regime improved survival of B16 HHDII/DR1 Luc1 tumour bearing mice when compared to dual vaccinated mice receiving PD-1 isotype control antibody and control sham vaccinated mice that received PD-1 isotype antibody. Analysis of tumour infiltrating lymphocyte populations revealed that dual vaccination increases CD8+ T-cell infiltrate into these intracranial tumours with these cells showing increased cell surface expression of the activation markers CD25 and CD69.

The dipeptide carnosine was also used to treat GBM cells in vitro, this molecule has previously been shown to have anti-tumour activity. When carnosine was used to treat GBM cells it led to reduced mitochondrial metabolism and migration of these cells. These properties make carnosine an attractive adjunct to immunotherapy.

Overall these results provide promise for the use of ImmunoBody® vaccination with immune checkpoint blockade for the treatment of GBM. Whilst immune cells can actively access tumours systemically administered checkpoint antibodies don't cross the blood-brain-barrier freely, therefore in order for these therapies to be further developed methods for improving brain penetrance of checkpoint inhibitors needs to be explored.

Item Type:	Thesis
Creators:	Pearson, J.R.D.
Date:	October 2019
Rights:	This work is the intellectual property of the author. You may copy up to 5 % of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed to the owner of the Intellectual Property Rights.
Additional Information:	This research was carried out in collaboration with the University of Portsmouth.
Divisions:	Schools > School of Science and Technology
Record created by:	Jeremy Silvester
Date Added:	27 Aug 2020 08:57
Last Modified:	31 May 2021 15:17
URI:	https://irep.ntu.ac.uk/id/eprint/40534