Identification of Immunogenic Neo-epitopes for the Development of Personalised Pancreatic Cancer Vaccines

Background Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumour types, with an extremely poor prognosis. Without active treatment, patients with metastatic PDAC have a mean survival of 3-5 months [1]. Current advances in surgical and adjuvant treatments have failed to improve overall survival rates since the 1970s. Thus, new treatment strategies, that are not cross resistant with conventional chemotherapy-based regimes are imperative. Recently, attention in cancer therapy has focused more heavily on immune-based strategies as these therapies act through a mechanism that is distinct from chemotherapy or radiation therapy and represent a non-cross-resistant treatment option [2]. Immune based therapies aim to stimulate robust T cell responses against tumour antigens. However, significant challenges exist in the development of these regimes. These include poor immunogenicity of the tumours and the presence of a highly immunosuppressive environment within the tumour [3, 4]. The clinical potential of various tumour vaccination strategies has been demonstrated in early phase clinical trials, with some promising immunological and clinical responses in PDAC patients [5-8]. A number of hurdles still need to be overcome in the development of an ideal PDAC vaccine. Crucially, specific tumour antigens must be identified that elicit a strong and specific immune response as failure of past cancer vaccine trials can be attributed in large part to selection of inappropriate tumour antigens that have weak inherent immune potential [9, 10]. Current advances in high throughput profiling technologies as enabled rapid determination of the genomic states of cancer cells such that comprehensive data regarding individual mutanomes is now available [11, 12]. It is also now possible to select missense mutations identified through the exome sequencing on the basis of their HLA binding capacity for production of synthetic peptides that can be presented by a desired HLA molecule [12, 13]. The development of this platform has allowed us to analysed published data from 100 PDAC patients [14] and establish a dataset containing high-affinity HLA-A2 and HLA-DP4 (the most abundant HLA class I and II molecules [15, 16]) and HLA-E*01:01 and HLA-E*01:03-restricted neo-epitopes for analysis as peptide vaccine candidates.

Hypothesis Sequence analysis has allowed us to develop a peptide library of neo-epitopes that are expressed at high frequency in patient populations and have high binding affinities compared to their wild-type counterpart to HLA-A2, HLA-DP4, HLA-E*01:01 or HLA-*01:03 molecules. We hypothesise that a number of these will be sufficiently immunogenic to stimulate a T cell interferon-γ (IFN-γ) response in vitro, that will translate to an in vivo anti-tumour response. Immunogenic neo-epitopes can then be combined in a peptide vaccination program using adjuvants such as oncolytic viruses for targeted delivery and expression within tumours of PDAC patients to stimulate robust and long-term anti-tumour responses.

Aim The initial aim of this project is to perform in vitro validation of neo-epitope candidates selected from available mutanome data to determine their immunogenicity using peripheral blood mononuclear cells (PBMCs) from healthy individuals.

Research Plan PBMC samples from healthy individuals will be HLA typed using commercially available reagents from thermofisher scientific. HLA-A2, HLA-DP4, HLA-E*01:01 and/or HLA-E*01:03 positive samples will be pulsed with peptides selected after bioinformatics analysis of available sequence data. IFN-γ and interleukin-2 (IL-2) production by the T cells in the samples will be evaluated by ELISA after two rounds of stimulation within a two weeks time as a measure of peptide immunogenicity. Once immunogenic peptides have been identified, their wild-type counterparts will be analysed in parallel to confirm specificity for the mutated epitope. Immunogenic peptides whose wild-type counterparts do not elicit immune responses will then be selected for inclusion in an oncolytic virus-based vaccine to be analysed in vivo using transgenic HLA-A2/HLA-DP4 mice [17].