Browsing by Subject "immunopeptidome"

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  • Mäki, Toni (Helsingin yliopisto, 2020)
    The human immune system can provide a powerful tool in developing therapies against various cancers. Even though the idea of an immune system actively searching for and disposing of potential mutated tumor cells is over a century old, only recent developments in various fields such as mass spectrometry, immuno-checkpoint blockade strategies and in silico modelling have enabled the realization of the full potential of recruiting immune system to fight cancer and the possibilities of personalized therapies. These therapeutic methods, including but not limited to oncolytic virus therapies, T-cell therapies and cancer vaccines, are based on the body’s ability to recognize mutated antigen peptides presented on the cell surface by MCH-receptors (also known as HLA-receptors in humans) and the disposal of the malignant cells by cytotoxic T-cells. Thus, the capability to map the individual HLA-presented peptidome and differentiate the immunogenic peptides is a foundation for this plethora of therapies and is in focus of ongoing research. This master thesis is a part of a project aiming to set up immunoaffinity-purification/MS based method in order to analyse the ligandome and determine T-cell recognized cancer associated antigens from tumor cells. Objectives of the work: 1. Characterizing tumor cell lines. 2. Immunological assay set up. 3. Collecting cell culture material for the ligandome affinity purification. 4. In silico prediction if the immunogenicity of selected peptides and assessing their source proteins. Methods used: 1. Cell culture. 2. FACS-analysis. 3. MTS-viability assay. 4. Immunological assays (ELISA, ELISPOT). 5. Immunological bioinformatics analysis tools (IEDB) and database search (UniPROT). Results: 1. Flow cytometric analysis provided essential information of the cell line HLA-1 expression. Additional information of PD-L1 expression can be used to evaluate cell line’s immune-evasion abilities. Preliminary MTS assay is used to determine linear range and optimal time frame for the PBMC/cancer cell co-culture killing assay. 2. Interferon γ cytokine secretion was determined by ELISPOT to assess PBMC response against known antigens in a preliminary experiment to approximate usable range for the following antigen specific PBMC assays. ELISA is used to confirm the presence of HLA-I receptors in the ligandome affinity purification eluates and to estimate the efficacy of purification. 3. Feasibility of in silico methods in the prediction of immunogenic peptides was explored. The experiments provided information that can be applied to the further development of the immune ligandome discovery project. In silico methods were successfully used to characterize previously identified HLA-restricted peptides and one previously identified immunogenic T-cell epitope. Even if the data acquired in silico can be considered only nominally verified at this stage, the results are encouraging.
  • Peltonen, Karita; Feola, Sara; Umer, Husen M.; Chiaro, Jacopo; Mermelekas, Georgios; Ylösmaki, Erkko; Pesonen, Sari; Branca, Rui M. M.; Lehtiö, Janne; Cerullo, Vincenzo (2021)
    Simple Summary Immunotherapy has revolutionized cancer treatment, yet many tumors remain resistant to current immuno-oncology therapies. Here we explore a novel, customized oncolytic adenovirus vaccine platform as immunotherapy in a resistant tumor model. We present a workflow for customizing the oncolytic vaccine for improved tumor targeting. This targeting is based on experimentally discovered tumor antigens, which are incorporated as active components of the vaccine formulation. The pipeline may be further applied for designing personalized therapeutic cancer vaccines. Knowledge of clinically targetable tumor antigens is becoming vital for broader design and utility of therapeutic cancer vaccines. This information is obtained reliably by directly interrogating the MHC-I presented peptide ligands, the immunopeptidome, with state-of-the-art mass spectrometry. Our manuscript describes direct identification of novel tumor antigens for an aggressive triple-negative breast cancer model. Immunopeptidome profiling revealed 2481 unique antigens, among them a novel ERV antigen originating from an endogenous retrovirus element. The clinical benefit and tumor control potential of the identified tumor antigens and ERV antigen were studied in a preclinical model using two vaccine platforms and therapeutic settings. Prominent control of established tumors was achieved using an oncolytic adenovirus platform designed for flexible and specific tumor targeting, namely PeptiCRAd. Our study presents a pipeline integrating immunopeptidome analysis-driven antigen discovery with a therapeutic cancer vaccine platform for improved personalized oncolytic immunotherapy.
  • Feola, Sara; Chiaro, Jacopo; Martins, Beatriz; Cerullo, Vincenzo (2020)
    According to the latest available data, cancer is the second leading cause of death, highlighting the need for novel cancer therapeutic approaches. In this context, immunotherapy is emerging as a reliable first-line treatment for many cancers, particularly metastatic melanoma. Indeed, cancer immunotherapy has attracted great interest following the recent clinical approval of antibodies targeting immune checkpoint molecules, such as PD-1, PD-L1, and CTLA-4, that release the brakes of the immune system, thus reviving a field otherwise poorly explored. Cancer immunotherapy mainly relies on the generation and stimulation of cytotoxic CD8 T lymphocytes (CTLs) within the tumor microenvironment (TME), priming T cells and establishing efficient and durable anti-tumor immunity. Therefore, there is a clear need to define and identify immunogenic T cell epitopes to use in therapeutic cancer vaccines. Naturally presented antigens in the human leucocyte antigen-1 (HLA-I) complex on the tumor surface are the main protagonists in evocating a specific anti-tumor CD8+ T cell response. However, the methodologies for their identification have been a major bottleneck for their reliable characterization. Consequently, the field of antigen discovery has yet to improve. The current review is intended to define what are today known as tumor antigens, with a main focus on CTL antigenic peptides. We also review the techniques developed and employed to date for antigen discovery, exploring both the direct elution of HLA-I peptides and the in silico prediction of epitopes. Finally, the last part of the review analyses the future challenges and direction of the antigen discovery field.