Autologous dendritic cells and mRNA in cancer immunotherapy

Cancer is one of the most pressing health problems all over the world. It is expected that 25% of the European population will get cancer before the age of 75. Despite effective treatment in some cases of cancer with the conventional treatment modalities including chemotherapy, radiation therapy and surgery, too many people still relapse and eventually die of cancer. Therefore additional approaches are necessary to increase survival rate. Immunotherapy became a fourth treatment modality and has become a major weapon in the fight against cancer. Active immunotherapy tries to modulate the patient's own immune system in the fight against cancer and aims at inducing a memory and long-lasting effect, in contrast to the effect of chemo- and radiation therapy, that can be temporary and can result in drug resistance. Dendritic cells (DCs) are key players in orchestrating this immune response and by optimizing the dendritic cell-based therapies we can attack the tumor in the most efficient way. Understanding the immune responses initiated by the DCs will result in the generation of optimized antigen presenting dendritic cell therapies and eventually in disease regression, the ultimate goal of all cancer treatments. In this thesis we focused on 2 aspects of cancer immunotherapy. The first focus is the immune monitoring in peripheral blood of stage III and IV melanoma patients after receiving TriMix-DC therapy (mRNA encoding CD70, CD40L and a constitutively active TLR4 (caTLR4)), injected intradermally and/or intravenously. These responses were compared with the responses detected in delayed type hypersensitivity (DTH) skin biopsies. On the level of the tumor antigens, we were able to show a comparable response in both compartments, although on the level of antigen fine-specificity, a partial compartmentalization could be demonstrated. The immune response of one of the patients included in this study was monitored into more detail. This patient received TriMix-DCs via the combined intradermal and intravenous route. The results show that both in the peripheral blood and DTH skin biopsies broad T cell responses could be induced by treatment with TriMix-DC. This broad T cell response was coupled with a stable disease for almost 3 years, demonstrating the effectiveness of our TriMix-DC therapy. The second focus of this thesis was the optimization of a DC-cell based cancer treatment by modification of an mRNA-encoding Wilms' Tumor 1 (WT1) construct. The use of mRNA in cancer immunotherapy holds great promise due to its favorable characteristics: it does not integrate into the genome of the host, is safe and easy to produce. In addition mRNA can genetically be modified in order to achieve an improved translation and immunogenicity. This was achieved by a stepwise-approach including four different modifications. The first modification was the linking of the WT1 sequence with the lysosomal targeting signal of DC-LAMP (lysosome-associated-membrane-protein) to induce both CD4+ and CD8+ T cell responses. Secondly, the nuclear localization sequence from the WT1 sequence was removed to enhance cytoplasmic expression favoring major histocompatibility complex (MHC) presentation. The third modification was the in silico gene optimization for optimal codon usage and G/C content and removal of factors that are known to affect translational efficacy. Finally, this entire sequence was subcloned into another RNA transcription vector, the pST1-vector. We could show that by using this modified WT1 mRNA-encoding construct we were able to enhance antigen expression in vitro and immunogenicity in an in vivo m