Harnessing T cell immunity for the prevention and treatment of liver cancer

Hepatocellular carcinoma (HCC) accounts for more than 80% of all diagnosed cases of liver cancer which is a major cause of cancer related fatalities worldwide. With few improvements in the survival rates during the last decades, prevention of HCC is key in reducing its burden globally. Infection with hepatitis B virus (HBV) remains the main etiological risk factor for developing HCC whilst in HBV patients co-infected with hepatitis D virus (HDV), the risk of developing HCC is triplicated due to the accelerated liver disease progression. In studies I and II, we aimed to develop a therapeutic vaccine for chronic HBV and HDV, as a preventive strategy for HCC. In study III, we sought to unlock novel T cell-based immunotherapies, as treatment for advanced HCC through isolation of neoantigen-driven T cell receptors (TCRs). In study I, we show that a homologous preS1-HDAg DNA-based vaccine strategy was able to elicit robust T cell responses to HBV and HDV antigens and entryinhibiting antibodies that could limit HBV monoinfection in liver-humanized mice. In study II, a heterologous DNA prime and protein boost preS1-HDAg vaccine strategy improved immunogenicity and could circumvent the HBV-induced tolerance present in the chronically infected host. Additionally, vaccine-induced antibodies protected liver-humanized mice against a chronic HBV/HDV coinfection and importantly they could protect HBV infected human-liver mice from HDV superinfection. In study III, we studied the cancer-specific T cell responses in patients with HCC, and we could detect T cell reactivity against mutated neoantigens in 4 out of 7 screened HCC patients. We isolated (putative) tumorreactive TCRs for further evaluation of their expression and specificity. Neoantigen-specific TCRs could be utilized to genetically redirect a substantial quantity of T cells against tumor cells, thus offering a potential new treatment for advanced HCC. Taken together, as we continue to unravel the dynamics of the immune system and refine therapies in the context of chronic diseases, this thesis illuminates two promising T cell avenues in the form of active and passive T cell immunotherapy and provides novel insights in the development of preventive and therapeutic tools aiming at combatting liver cancer

Liquid Biopsy in Hepatocellular Carcinoma: Opportunities and Challenges for Immunotherapy

Hepatocellular carcinoma (HCC) is one of the deadliest cancer types worldwide. HCC is often diagnosed at a late stage when the therapeutic options are very limited. However, even at the earlier stages, the best treatment is liver transplantation, surgical resection or ablation. Surgical resection and ablation may carry a high risk of tumor recurrence. The recent introduction of immunotherapies resulted in clinical responses for a subgroup of patients, but there were still no effective predictive markers for response to immunotherapy or for recurrence after surgical therapy. The identification of biomarkers that could correlate and predict response or recurrence would require close monitoring of the patients throughout and after the completion of treatment. However, this would not be performed efficiently by repeated and invasive tissue biopsies. A better approach would be to use liquid biopsies including circulating tumor DNA (ctDNA), circulating RNA (e.g., microRNAs), circulating tumor cells (CTC) and extracellular vesicles (EVs) (e.g., exosomes) for disease monitoring in a non-invasive manner. In this review, we discuss the currently available technology that can enable the use of liquid biopsy as a diagnostic and prognostic tool. Moreover, we discuss the opportunities and challenges of the clinical application of liquid biopsy for immunotherapy of HCC

PD-1 expression affects cytokine production by ILC2 and is influenced by peroxisome proliferator-activated receptor-γ.

Innate lymphoid cells (ILCs) can provide early cytokine help against a variety of pathogens in the lungs and gastrointestinal tract. Type 2 ILC (ILC2) are comparable to T helper 2 cells found in the adaptive immune system, which secrete cytokines such as interleukin 5 (IL-5) and IL-13 and have been found to play roles in host defense against helminth infections and in allergic responses. Recent studies have identified that programmed cell death protein 1 (PD-1) and peroxisome proliferator activated receptor-γ (PPAR-γ) are highly expressed by ILC2. We examined whether PD-1 plays a role in ILC2 function and whether there was any connection between PD-1 and PPAR-γ METHODS: To ensure that only innate immune cells were present, ILC2 cells were examined from RAG1-/- and PD-1-/- xRAG1-/- mice under steady-state or following inoculation with IL-33. We also tested ILC2 generated from bone marrow of RAG1-/- and PD-1-/- xRAG1-/- mice for their production of cytokines. These in vitro-derived ILC2 were also exposed to agonist and antagonist of PPAR-γ

Novel prime-boost immune-based therapy inhibiting both hepatitis B and D virus infections

Objective Chronic HBV/HDV infections are a major cause of liver cancer. Current treatments can only rarely eliminate HBV and HDV. Our previously developed preS1-HDAg immunotherapy could induce neutralising antibodies to HBV in vivo and raise HBV/HDV-specific T-cells. Here, we further investigate if a heterologous prime-boost strategy can circumvent T-cell tolerance and preclude HDV superinfection in vivo. Design A DNA prime-protein boost strategy was evaluated for immunogenicity in mice and rabbits. Its ability to circumvent T-cell tolerance was assessed in immunocompetent hepatitis B surface antigen (HBsAg)-transgenic mice. Neutralisation of HBV and HDV was evaluated both in vitro and in immunodeficient human-liver chimeric mice upon adoptive transfer. Results The prime-boost strategy elicits robust HBV/HDV-specific T-cells and preS1-antibodies that can effectively prevent HBV and HDV (co-)infection in vitro and in vivo. In a mouse model representing the chronic HBsAg carrier state, active immunisation primes high levels of preS1-antibodies and HDAg-specific T-cells. Moreover, transfer of vaccine-induced antibodies completely protects HBV-infected human-liver chimeric mice from HDV superinfection. Conclusion The herein described preS1-HDAg immunotherapy is shown to be immunogenic and vaccine-induced antibodies are highly effective at preventing HBV and HDV (super)infection both in vitro and in vivo. Our vaccine can complement current and future therapies for the control of chronic HBV and HDV infection

Process Development for Adoptive Cell Therapy in Academia: A Pipeline for Clinical-Scale Manufacturing of Multiple TCR-T Cell Products

Cellular immunotherapies based on T cell receptor (TCR) transfer are promising approaches for the treatment of cancer and chronic viral infections. The discovery of novel receptors is expanding considerably; however, the clinical development of TCR-T cell therapies still lags. Here we provide a pipeline for process development and clinical-scale manufacturing of TCR-T cells in academia. We utilized two TCRs specific for hepatitis C virus (HCV) as models because of their marked differences in avidity and functional profile in TCR-redirected cells. With our clinical-scale pipeline, we reproduced the functional profile associated with each TCR. Moreover, the two TCR-T cell products demonstrated similar yield, purity, transduction efficiency as well as phenotype. The TCR-T cell products had a highly reproducible yield of over 1.4 × 109 cells, with an average viability of 93%; 97.8-99% of cells were CD3+, of which 47.66 ± 2.02% were CD8+ T cells; the phenotype was markedly associated with central memory (CD62L+CD45RO+) for CD4+ (93.70 ± 5.23%) and CD8+ (94.26 ± 4.04%). The functional assessments in 2D and 3D cell culture assays showed that TCR-T cells mounted a polyfunctional response to the cognate HCV peptide target in tumor cell lines, including killing. Collectively, we report a solid strategy for the efficient large-scale manufacturing of TCR-T cells