HAGE and WT1 proteins as promising immunotherapeutic targets in chronic myeloid leukaemia

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Almshayakhchi, R.N.S., 2020. HAGE and WT1 proteins as promising immunotherapeutic targets in chronic myeloid leukaemia. PhD, Nottingham Trent University.

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Abstract

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder resulting from a malignant transformation of the primitive HSCs. The disease is characterised by the breaking and re-assembling of chromosomes 9 and 22 t(9;22)(q34;q11) and the formation of a fusion gene called BCR-ABL1 and its chimeric oncogenic protein (p210) which results in a permanent activation of tyrosine kinase (TK) enzyme. The clinical outcome of CML has significantly improved since the discovery of tyrosine kinase inhibitors (TKIs); however, they are not curative, and resistance against these therapies could develop at any phase of the disease. The molecular characteristic of CML pathology, the nature of cancerous-host immune cells interaction, in addition to the beneficial immunomodulatory role of the TKI therapy, would all offer "in theory" an excellent opportunity to combine TKIs with immunotherapeutic strategies. The cancer testis antigen DDX43 (HAGE) and the oncogene Wilms' tumour (WT1) antigens have previously been shown to be immunogenic and overexpressed in CML at mRNA level at 57-71% and 50-100%, respectively. With the aim to help patients who are on imatinib therapy to achieve a "definitive cure" by eliminating residual CML cells, patients who developed resistance to imatinib at any phase of the disease, and patients who are in blast crisis and not eligible to allo-HSCT, it was, therefore, hypothesised that a combination of HAGE and WT1 vaccines will be more effective against established CML tumours expressing both antigens than a vaccine incorporating peptides derived from either of these antigens alone.

This project investigated the use of HAGE- and WT1-derived ImmunBody® vaccines, alone and together in generating vaccine specific release of cytokines and in specifically killing target cells expressing the relevant antigens and HLA-A haplotype by the vaccine induced T cells. In order to achieve this, HHDII/DR1 mice were immunised with HAGE- and/or WT1-ImmunoBody® bullets of DNA vaccines in a prime-boost regime in two different flanks using gene gun technology. The immune responses generated against these two antigens were then assessed by measuring the number of IFN- producing cells using ELISpot assay, cytokines released by flow cytometry and killing against specific targets using Chromium release assay. A number of CML-derived targets were genetically modified to either express or suppress the expression of HAGE/WT1 proteins, and the success of these modifications was confirmed by RT-qPCR and Western blot assays. Results showed that while both vaccines were independently capable of inducing the release of IFN- in a high proportion of vaccine-induced cells and provoking substantial cellmediated killing, the combined vaccines approach was able to induce a significantly higher number of CD8+ T cells producing TNF- and a significantly higher percentage of target cell death in comparison with the individual vaccines.

More importantly, in tumour challenges models, "humanised" B16 (HAGE+/WT1+) cells transplanted subcutaneously were employed as a "proof-of-concept" to demonstrate in vivo vaccine efficacy. The combination of HAGE/WT1 ImmunoBody® vaccines was shown to significantly delay tumour growth and prolong mice survival in a prophylactic setting in comparison to mice that had not received the vaccines or even mice that had received only one of the vaccines. Overall, this work has demonstrated the significant value of combining both HAGE- and WT1-ImmunoBody® vaccines for treating patients harbouring HAGE+/WT+ cancerous cells.

Item Type:	Thesis
Description:	Abridged version.
Creators:	Almshayakhchi, R.N.S.
Date:	May 2020
Rights:	This work is the intellectual property of the author and may also be owned by the research sponsor(s) and/or Nottingham Trent University. 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 in the owner(s) of the Intellectual Property Rights.
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	13 Aug 2020 12:47
Last Modified:	28 May 2021 03:00
URI:	https://irep.ntu.ac.uk/id/eprint/40449